Microparticles manufactured in an oil-in-water process comprising a prostamide

ABSTRACT

Biocompatible microparticles include an ophthalmically active cyclic lipid component and a biodegradable polymer that is effective, when placed into the subconjunctival space, in facilitating release of the cyclic lipid component into the anterior and posterior segments of an eye for an extended period of time. The cyclic lipid component can be associated with a biodegradable polymer matrix, such as a matrix of a two biodegradable polymers. Or, the cyclic lipid component can be encapsulated by the polymeric component. The present microparticles include oil-in-water emulsified microparticles. The subconjunctivally administered microparticles can be used to treat or to reduce at least one symptom of an ocular condition, such as glaucoma or age related macular degeneration.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 11/371,118 filed Mar. 8, 2006 which is a continuation-in-partof the U.S. patent application entitled “METHODS FOR TREATING OCULARCONDITIONS WITH CYCLIC LIPID CONTAINING MICROPARTICLES” of which theco-inventors are Patrick Hughes, Joan-En Lin and Devin Welty, filed withthe United States Patent and Trademark Office on Mar. 6, 2006, serialnumber pending, which is a continuation-in-part of U.S. application Ser.No. 11/303,462, filed Dec. 15, 2005 which is a continuation-in-part ofapplication Ser. No. 10/837,260, filed Apr. 30, 2004, the entirecontents of which prior applications are hereby incorporated byreference.

BACKGROUND

The present invention generally relates to methods for treating an eyeof a patient, and more specifically to methods comprisingsubconjunctival administration microparticles to an eye that providetherapeutic agents, such as ophthalmically active cyclic lipidcomponents, to an eye in which the microparticles are placed.

Ocular hypotensive agents are useful in the treatment of a number ofvarious ocular hypertensive conditions, such as post-surgical andpost-laser trabeculectomy ocular hypertensive episodes, glaucoma, and aspresurgical adjuncts.

Glaucoma is a disease of the eye characterized by increased intraocularpressure. On the basis of its etiology, glaucoma has been classified asprimary or secondary. For example, primary glaucoma in adults(congenital glaucoma) may be either open-angle or acute or chronicangle-closure. Secondary glaucoma results from pre-existing oculardiseases such as uveitis, intraocular tumor or an enlarged cataract.

The underlying causes of primary glaucoma are not yet known. Theincreased intraocular tension is due to the obstruction of aqueous humoroutflow. In chronic open-angle glaucoma, the anterior chamber and itsanatomic structures appear normal, but drainage of the aqueous humor isimpeded. In acute or chronic angle-closure glaucoma, the anteriorchamber is shallow, the filtration angle is narrowed, and the iris mayobstruct the trabecular meshwork at the entrance of the canal ofSchlemm. Dilation of the pupil may push the root of the iris forwardagainst the angle, and may produce pupillary block and thus precipitatean acute attack. Eyes with narrow anterior chamber angles arepredisposed to acute angle-closure glaucoma attacks of various degreesof severity.

Secondary glaucoma is caused by any interference with the flow ofaqueous humor from the posterior chamber into the anterior chamber andsubsequently, into the canal of Schlemm. Inflammatory disease of theanterior segment may prevent aqueous escape by causing completeposterior synechia in iris bombe and may plug the drainage channel withexudates. Other common causes are intraocular tumors, enlargedcataracts, central retinal vein occlusion, trauma to the eye, operativeprocedures and intraocular hemorrhage.

Considering all types together, glaucoma occurs in about 2% of allpersons over the age of 40 and may be asymptotic for years beforeprogressing to rapid loss of vision. In cases where surgery is notindicated, topical beta-adrenoreceptor antagonists have traditionallybeen the drugs of choice for treating glaucoma.

Prostaglandins were earlier regarded as potent ocular hypertensives;however, evidence accumulated in the last two decades shows that someprostaglandins are highly effective ocular hypotensive agents and areideally suited for the long-term medical management of glaucoma. (See,for example, Starr, M. S. Exp. Eye Res. 1971, 11, pp. 170-177; Bito, L.Z. Biological Protection with Prostaglandins Cohen, M. M., ed., BocaRaton, Fla., CRC Press Inc., 1985, pp. 231-252; and Bito, L. Z., AppliedPharmacology in the Medical Treatment of Glaucomas Drance, S. M. andNeufeld, A. H. eds., New York, Grune & Stratton, 1984, pp. 477-505).Such prostaglandins include PGF_(2α), PGF_(1α), PGE₂, and certainlipid-soluble esters, such as C₁ to C₅ alkyl esters, e.g. 1-isopropylester, of such compounds.

In U.S. Pat. No. 4,599,353 certain prostaglandins, in particular PGE₂and PGF_(2α) and the C₁ to C₅ alkyl esters of the latter compound, werereported to possess ocular hypotensive activity and were recommended foruse in glaucoma management.

Although the precise mechanism is not yet known, recent experimentalresults indicate that the prostaglandin-induced reduction in intraocularpressure results from increased uveoscleral outflow [Nilsson et al.,Invest. Ophthalmol. Vis. Sci. 28(suppl), 284 (1987)].

The isopropyl ester of PGF_(2α) has been shown to have significantlygreater hypotensive potency than the parent compound, which wasattributed to its more effective penetration through the cornea. In1987, this compound was described as “the most potent ocular hypotensiveagent ever reported.” [See, for example, Bito, L. Z., Arch. Ophthalmol.105, 1036 (1987), and Siebold et al., Prodrug 5, 3 (1989)].

Whereas prostaglandins appear to be devoid of significant intraocularside effects, ocular surface (conjunctival) hyperemia and foreign-bodysensation have been consistently associated with the topical ocular useof such compounds, in particular PGF_(2α) and its prodrugs, e.g. its1-isopropyl ester, in humans. The clinical potential of prostaglandinsin the management of conditions associated with increased ocularpressure, e.g. glaucoma, is greatly limited by these side effects.

Certain prostaglandins and their analogs and derivatives, such as thePGF_(2α) derivative latanoprost, sold under the trademark Xalatan®, havebeen established as compounds useful in treating ocular hypertension andglaucoma. However, latanoprost, the first prostaglandin approved by theUnited States Food And Drug Administration for this indication, is aprostaglandin derivative possessing the undesirable side effect ofproducing an increase in brown pigment in the iris of 5-15% of humaneyes. The change in color results from an increased number ofmelanosomes (pigment granules) within iridial melanocytes. See e.g.,Watson et al., Ophthalmology 103:126 (1996). While it is still unclearwhether this effect has additional and deleterious clinicalramifications, from a cosmetic standpoint alone such side effects areundesirable. Certain phenyl and phenoxy mono, tri and tetraprostaglandins and their 1-esters are disclosed in European PatentApplication 0,364,417 as useful in the treatment of glaucoma or ocularhypertension.

In a series of United States patent applications assigned to Allergan,Inc. prostaglandin esters with increased ocular hypotensive activityaccompanied with no or substantially reduced side-effects are disclosed.U.S. patent application Ser. No. 386,835 (filed Jul. 27, 1989), relatesto certain 11-acyl-prostaglandins, such as 11-pivaloyl, 11-acetyl,11-isobutyryl, 11-valeryl, and 11-isovaleryl PGF_(2α). Intraocularpressure reducing 15-acyl prostaglandins are disclosed in U.S. Ser. No.357,394 (filed May 25, 1989). Similarly, 11,15-9,15- and 9,11-diestersof prostaglandins, for example 11,15-dipivaloyl PGF_(2α) are known tohave ocular hypotensive activity. See U.S. Ser. No. 385,645 filed Jul.27, 1990, now U.S. Pat. No. 4,494,274; Ser. No. 584,370 which is acontinuation of U.S. Ser. No. 386,312, and U.S. Ser. No. 585,284, nowU.S. Pat. No. 5,034,413 which is a continuation of U.S. Ser. No.386,834, where the parent applications were filed on Jul. 27, 1989.

Woodward et al U.S. Pat. Nos. 5,688,819 and 6,403,649 disclose certaincyclopentane heptanoic acid, 2-cycloalkyl or arylalkyl compounds asocular hypotensives. These compounds, which can properly becharacterized as hypotensive lipids, are effective in treating ocularhypertension.

As one example, the prostamide analog, bimatoprost, has been discoveredto be effective in reducing intraocular pressure possibly by increasingthe aqueous humour outflow of an eye (Woodward et al., AGN 192024(Lumigan®): A Synthetic Prostamide Analog that Lowers PrimateIntraocular Pressure by Virtue of Its Inherent Pharmacological Activity,ARVO 2002; (CD-ROM):POS; Chen et al., Lumigan®: A Novel Drug forGlaucoma Therapy, Optom In Pract, 3:95-102 (2002); Coleman et al., A3-Month Randomized Controlled Trial of Bimatoprost (LUMIGAN) versusCombined Timolol and Dorzolamide (Cosopt) in Patients with Glaucoma orOcular Hypertension, Ophthalmology 110(12): 2362-8 (2003); Brubaker,Mechanism of Action of Bimatoprost (Lumigan™), Surv Ophthalmol 45 (Suppl4):S347-S351 (2001); and Woodward et al., The Pharmacology ofBimatoprost (Lumigan™), Surv Ophthalmol 45 (Suppl 4) S337-S345 (2001).

Bimatoprost is an analog (e.g., a structural derivative) of a naturallyoccurring prostamide. Bimatoprost's chemical name is(Z)-7-[(1R,2R,3R,5S)-3,5-Dihydroxy-2-[1E,3S)-3-hydroxy-5-phenyl-1-pentenyl]cyclopentyl]-5-N-ethylheptenamide,and it has a molecular weight of 415.58. Its molecular formula isC₂₅H₃₇NO₄. Bimatoprost is available in a topical ophthalmic solutionunder the tradename Lumigan® (Allergan, Inc.). Each mL of the solutioncontains 0.3 mg of bimatoprost as the active agent, 0.05 mg ofbenzalkonium chloride (BAK) as a preservative, and sodium chloride,sodium phosphate, dibasic; citric acid; and purified water as inactiveagents.

Biocompatible implants for placement in the eye have been disclosed in anumber of patents, such as U.S. Pat. Nos. 4,521,210; 4,853,224;4,997,652; 5,164,188; 5,443,505; 5,501,856; 5,766,242; 5,824,072;5,869,079; 6,074,661; 6,331,313; 6,369,116; and 6,699,493.

Yamada et al. US Patent Publication 2006/0013859 discuss thesubconjunctival injection of gels or liquids capable of forming gels insitu in which a sparingly soluble drug-containing component iscontained, in order to form a depot for ocular drug delivery, includingdelivery of such drugs to the posterior segment of the eye.

It would be advantageous to provide methods using eye implantable drugdelivery systems, such as microparticles that are capable of releasing atherapeutic agent, such as a cyclic lipid component, preferably at asustained or controlled rate for extended periods of time and in amountswith few or no negative side effects. In those cases in which it mayprovide a possible alternative to topical drug delivery, such a systemwould automatically release the drug over a period of time, thusreducing concerns with daily patient compliance.

SUMMARY

The present invention provides new drug delivery methods for drugrelease, advantageously extended and/or sustained and/or controlled drugrelease, into an eye, for example, to achieve one or more desiredtherapeutic effects. The present methods employ drug delivery systems inthe form of microparticles that are administered subconjuctivally to aneye. The present methods advantageously provide for extended releasetimes of one or more therapeutic agents, such as one or moreophthalmically active cyclic lipid components. Thus, the patient inwhose eye the microparticles have been placed receives a therapeuticamount of an agent or agents for a relatively long or extended timeperiod without requiring additional administrations of the agent oragents. For example, the patient has a therapeutically active agentavailable for treatment of the eye over a relatively long period oftime, for example, on the order of at least about one week, such asbetween about two and about six months after administering themicroparticles. Such extended release times facilitate obtainingsuccessful treatment results. In addition, administering suchmicroparticles subconjunctivally preferably reduces the occurrenceand/or severity of at least one side effect, for example, hyperemia,relative to administering an identical amount of the cyclic lipidcomponent to the eye in the form of a topical composition. Further,subconjunctival administration of microparticles comprising cyclic lipidcomponents has unexpectedly been found to be highly effective inproviding such cyclic lipid components to the retina of the eye. InLaedwif M. S. et al., PROSTAGLANDINS LEUKOT. ESSENT. FATTY ACIDS72:251-6 (April 2005), hereby incorporated by reference herein, a studyshowed infusion treatment of patients suffering from age-related maculardegeneration (ARMD), particularly dry ARMD, with a cyclic lipid(prostaglandin E1) results in an improvement in visual acuity in 8/11patients two months after the end of the infusion period. As thesubconjunctival administration of microspheres containing a cyclic lipidcomponent results in particularly effective delivery of such agents tothe retina, the present invention would provide a particularlyadvantageous method of delivering the drug to ocular tissue without thepossibility of side effects which may occur in the systemicadministration of certain such cyclic lipids.

Microparticles in accordance with the disclosure herein comprise atherapeutic component and a drug release sustaining component associatedwith the therapeutic component. In accordance with the presentinvention, the therapeutic component comprises, consists essentially of,or consists of, a cyclic lipid component, such as, without limitation, aprostaglandin, prostaglandin analog, prostaglandin derivative,prostamide, prostamide analog, and a prostamide derivative that iseffective in providing an ophthalmic therapeutic effect, such as,without limitation, reducing or maintaining a reduced intraocularpressure in a hypertensive eye, or providing to the retina of an eye aneffective amount of a cyclic lipid component having neuroprotectiveactivities. The microparticles are associated with the therapeuticcomponent to sustain release of an amount of the cyclic lipid componentinto an eye in which the microparticles are placed. The cyclic lipidcomponent is released into the eye for an extended period of time afterthe microparticles are administered subconjunctivally and is effectivein treating or reducing at least one symptom of an ocular condition ofan eye. Advantageously, the present microparticles may be effective inrelieving a hypertensive eye by reducing the intraocular pressure of theeye or maintaining the intraocular pressure at a reduced level withoutsubstantial amounts of ocular hyperemia. Alternatively, the presentmicroparticles may be effective in relieving disorders of the posteriorsegment of the eye, particularly, a retinal condition such as exudativeor non-exudative age-related macular degeneration, by delivering cycliclipid components via the sclera to the tissues of the posterior segment,in particular, the retina.

Embodiments of the present cyclic lipid-containing microparticles can beunderstood from the following description and claims.

In one embodiment, the microparticles comprise a cyclic lipid componentand a biodegradable polymer matrix. The cyclic lipid component isassociated with a biodegradable polymer matrix that releases drug at arate effective to sustain release of an amount of the cyclic lipidcomponent from the microparticles effective to treat an ocularcondition. The microparticles are biodegradable or bioerodible andprovide a sustained release of the cyclic lipid component to either orboth the anterior and posterior segments of the eye for extended periodsof time, such as for more than one week, for example for about threemonths or more and up to about six months or more.

The biodegradable polymer component of the foregoing microparticles maybe a mixture of biodegradable polymers, wherein at least one of thebiodegradable polymers is a polylactic acid polymer having a molecularweight less than 64 kiloDaltons (kD). Additionally or alternatively, theforegoing microparticles may comprise a first biodegradable polymer of apolylactic acid, and a different second biodegradable polymer of apolylactic acid. Furthermore, the foregoing microparticles may comprisea mixture of different biodegradable polymers, each biodegradablepolymer having an inherent viscosity in a range of about 0.2deciliters/gram (dl/g) to about 1.0 dl/g.

The cyclic lipid component of the implants disclosed herein may includea therapeutic component comprising a prostaglandin, prostaglandinanalog, prostaglandin derivative, prostamide, prostamide analog, or aprostamide derivative, that is effective in treating ocular conditions.One example of a suitable prostamide derivative is bimatoprost. Otherexamples of cyclic lipid components of the present invention include,without limitation, latanoprost, travoprost and unoprostone and saltsderivatives, and analogs of these. In addition, the therapeuticcomponent of the present microspheres may include one or more additionaland different therapeutic agents that may be effective in treating anocular condition.

A method of making the present microspheres involves combining or mixingthe cyclic lipid component with a biodegradable polymer or polymers. Themixture may then be extruded or compressed to form a single composition.The single composition may then be processed to form microspheressuitable for placement subconjunctivally.

A method of making the present microspheres may also include using anoil-in-oil emulsion process to form the microspheres. Such methods maybe particularly useful in forming microparticles, nanoparticles and thelike. Thus, an embodiment of the present invention relates to methods ofmaking microparticles using an oil-in-oil emulsion process andmicroparticles so produced, as described herein.

The microspheres, which may include a population of microparticles ornanoparticles, may be placed in an ocular region such as, withoutlimitation, subconjunctivally, to treat a variety of ocular conditionsof the anterior or posterior segment. For example, the microspheres maybe effective in delivering a therapeutic component comprising a cycliclipid to tissues of the anterior segment, thereby reducing ocularhypertension, and thus may be effective in reducing at least one symptomof an ocular condition associated with an increased intraocularpressure. Alternatively, subconjunctival administration if themicrospheres of the present invention are very effective at deliveringthe therapeutic component to the retina and other tissues of theposterior segment for the treatment of neurodegenerative conditions suchas age related macular degeneration (ARMD), such as “wet” or “dry” ARMD.

Kits in accordance with the present invention may comprise one or moreof the present microspheres, and instructions for using themicrospheres. For example, the instructions may explain how toadminister the microspheres to a patient, and types of conditions thatmay be treated with the microspheres.

The present invention also encompasses the use of the presentmicrospheres in treating a patient, such as in treating one or more ofthe conditions or diseases set forth herein, as well as medicaments,which are oil-in-oil emulsified microparticles, for treating an ocularcondition of a patient. The invention also encompasses the use of acyclic lipid component and a polymeric component, as described herein,in the manufacture of a medicament for treating a patient.

As an alternative to subconjunctival injection, the drug containingmicrospheres disclosed herein can be placed onto the surface of the eyeby injection through a hollow dome sutured onto the eye, as set forthfor example in U.S. patent application 2005 113806A1 and ininternational patent application WO 03/020172A1, to thereby prevent orreduce the leaching or wash out of the drug from the site of its ocularadministration.

Additional aspects and advantages of the present invention are set forthin the following description and claims, particularly when considered inconjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is flow chart of a method of making microspheres.

FIG. 2A is the chemical structure of bimatoprost.

FIG. 2B is the chemical structure of 15β bimatoprost.

FIG. 2C is the chemical structure of 5,6,-trans bimatoprost isomer.

FIG. 2D is the chemical structure of the C1 acid of bimatoprost.

FIG. 2E is the chemical structure of triphenyphosphine oxide.

FIG. 2F is the chemical structure of 15-Keto bimatoprost.

FIG. 3A is a chromatogram of a bimatoprost standard.

FIG. 3B is a chromatogram of non-sterilized bimatoprost microparticles.

FIG. 3C is a chromatogram of sterilized bimatoprost microparticles.

FIG. 3D is a chromatogram of non-sterilized placebo compositions.

FIG. 3E is a chromatogram of sterilized placebo compositions.

FIG. 4A is a graph of volume % as a function of particle diameter.

FIG. 4B is a graph of number % as a function of particle diameter.

FIG. 4C is a graph of volume % as a function of particle diameter for adifferent batch of microparticles than shown in FIG. 4A.

FIG. 4D is a graph of number % as a function of particle diameter forthe batch of FIG. 4C.

FIG. 5A is a photograph of one batch of sterile microspheres.

FIG. 5B is a photograph of a different batch of sterile microspheresother than the batch of FIG. 5A.

FIG. 5C is a photograph of non-sterile microspheres.

FIG. 5D is a photograph of sterile microspheres.

FIG. 5E is a photograph of a non-sterile batch of microspheres.

FIG. 5F is a photograph of sterile microspheres.

FIG. 6 is a graph percent bimatoprost released as a function of time.

FIG. 7 is a plot of the in vitro release of drug from irradiated andnon-irradiated microspheres.

DESCRIPTION

As described herein, controlled and sustained administration of atherapeutic agent through the subconjunctival administration of one ormore microparticles, may improve treatment of undesirable ocularconditions of the anterior or posterior segment. The microparticlescomprise a pharmaceutically acceptable polymeric composition and areformulated to release one or more pharmaceutically active agents, suchas a cyclic lipid, or other intraocular pressure lowering orneuroprotective agent, over an extended period of time. The microspheresare effective to provide a therapeutically effective dosage of the agentor agents to a region of the eye to treat or prevent one or moreundesirable ocular conditions. Thus, with a single administration,cyclic lipids will be made available at the site where they are neededand will be maintained for an extended period of time, rather thansubjecting the patient to repeated injections or repeated administrationof topical drops.

The microparticles of the present invention comprise a therapeuticcomponent and a drug release-sustaining component associated with thetherapeutic component. In accordance with the present invention, thetherapeutic component comprises, consists essentially of, or consistsof, a cyclic lipid component. The drug release sustaining component isassociated with the therapeutic component to sustain release of aneffective amount of the cyclic lipid component into an eye in which themicroparticles are placed. The amount of the cyclic lipid component isreleased into the eye for a period of time greater than about one weekafter the microparticles are placed in the eye, and is effective intreating or reducing a symptom of an ocular condition, such as ocularhypertension of retinal degeneration.

DEFINITIONS

For the purposes of this description, we use the following terms asdefined in this section, unless the context of the word indicates adifferent meaning.

As used herein, a “microsphere” or “microparticle” are interchangeablyused to refer to a device or element that is structured, sized, orotherwise configured to be administered subconjunctivally. It will beunderstood that the term microspheres or microparticles includesparticles, micro or nanospheres, small fragments, microparticles,nanoparticles, fine powders and the like comprising a biocompatiblematrix encapsulating or incorporating a therapeutic component.Microspheres are generally biocompatible with physiological conditionsof an eye and do not cause adverse side effects. Microspheresadministered subconjunctivally may be used safely without disruptingvision of the eye. Microspheres have a maximum dimension, such asdiameter or length, less than 1 mm. For example, microparticles can havea maximum dimension less than about 500 μm. Microparticles may also havea maximum dimension no greater than about 200 μm, or may have a maximumdimension from about 30 μm to about 50 μm, among other sizes.

As used herein, a “therapeutic component” refers to that portion of amicrosphere other than the polymer matrix comprising one or moretherapeutic agents or substances used to treat a medical condition ofthe eye. The therapeutic component may be a discrete region of amicrosphere, or it may be homogenously distributed throughout themicrosphere. The therapeutic agents of the therapeutic componentcomprise at least one cyclic lipid and are typically ophthalmicallyacceptable, and are provided in a form that does not cause significantadverse reactions when the microsphere is placed in an eye.

As used herein, a “cyclic lipid component” refers to a portion of anintraocular implant that comprises one or more cyclic lipids havingocular therapeutic activity, including, without limitation, aprostaglandin, prostaglandin analog, prostaglandin derivative,prostamide, prostamide analog, and a prostamide derivative that iseffective in providing an ophthalmic therapeutic effect, such as,without limitation, reducing or maintaining a reduced intraocularpressure in a hypertensive eye, or providing to the retina of an eye aneffective amount of a cyclic lipid component having neuroprotectiveactivities. Cyclic lipids having anti-glaucoma activity can beidentified by applying the cyclic lipid to an eye with increasedintraocular pressure, and evaluating whether the intraocular pressuredecreases after the application. Cyclic lipids having neuroprotectiveactivity may be identified by, for example, intravitreal administrationof the cyclic lipid to an eye having a neurodegenerative disorder suchas ARMD, and evaluating whether the neurodegeneration is slowed orhalted, or whether visual acuity has increased.

As used herein, a “drug release sustaining component” refers to aportion of the microsphere that is effective to provide a sustainedrelease of the therapeutic agents from the microsphere. A drug releasesustaining component may be a biodegradable polymer matrix, or it may bea coating covering a core region of the microsphere that comprises atherapeutic component.

As used herein, “associated with” means mixed with, dispersed within,coupled to, covering, or surrounding.

As used herein, an “ocular region” or “ocular site” refers generally toany area of the eyeball, including the anterior and posterior segment ofthe eye, and which generally includes, but is not limited to, anyfunctional (e.g., for vision) or structural tissues found in theeyeball, or tissues or cellular layers that partly or completely linethe interior or exterior of the eyeball. Specific examples of areas ofthe eyeball in an ocular region include the anterior chamber, theposterior chamber, the vitreous cavity, the choroid, the suprachoroidalspace, the conjunctiva, the subconjunctival space, the episcleral space,the intracorneal space, the epicorneal space, the sclera, the parsplana, surgically-induced avascular regions, the macula, and the retina.

As used herein, an “ocular condition” is a disease, ailment or conditionwhich affects or involves the eye or one of the parts or regions of theeye. Broadly speaking the eye includes the eyeball and the tissues andfluids which constitute the eyeball, the periocular muscles (such as theoblique and rectus muscles) and the portion of the optic nerve which iswithin or adjacent to the eyeball.

An anterior ocular condition is a disease, ailment or condition whichaffects or which involves an anterior (i.e. front of the eye) ocularregion or site, such as a periocular muscle, an eye lid or an eye balltissue or fluid which is located anterior to the posterior wall of thelens capsule or ciliary muscles. Thus, an anterior ocular conditionprimarily affects or involves the conjunctiva, the cornea, the anteriorchamber, the iris, the posterior chamber (behind the retina but in frontof the posterior wall of the lens capsule), the lens or the lens capsuleand blood vessels and nerve which vascularize or innervate an anteriorocular region or site.

Thus, an anterior ocular condition can include a disease, ailment orcondition, such as for example, aphakia; pseudophakia; astigmatism;blepharospasm; cataract; conjunctival diseases; conjunctivitis; cornealdiseases; corneal ulcer; dry eye syndromes; eyelid diseases; lacrimalapparatus diseases; lacrimal duct obstruction; myopia; presbyopia; pupildisorders; refractive disorders and strabismus. Glaucoma can also beconsidered to be an anterior ocular condition because a clinical goal ofglaucoma treatment can be to reduce a hypertension of aqueous fluid inthe anterior chamber of the eye (i.e. reduce intraocular pressure).

A posterior ocular condition is a disease, ailment or condition whichprimarily affects or involves a posterior ocular region or site such aschoroid or sclera (in a position posterior to a plane through theposterior wall of the lens capsule), vitreous, vitreous chamber, retina,optic nerve (i.e. the optic disc), and blood vessels and nerves whichvascularize or innervate a posterior ocular region or site.

Thus, a posterior ocular condition can include a disease, ailment orcondition, such as for example, acute macular neuroretinopathy; Behcet'sdisease; choroidal neovascularization; diabetic uveitis; histoplasmosis;infections, such as fungal or viral-caused infections; maculardegeneration, such as acute macular degeneration, non-exudative agerelated macular degeneration and exudative age related maculardegeneration; edema, such as macular edema, cystoid macular edema anddiabetic macular edema; multifocal choroiditis; ocular trauma whichaffects a posterior ocular site or location; ocular tumors; retinaldisorders, such as central retinal vein occlusion, diabetic retinopathy(including proliferative diabetic retinopathy), proliferativevitreoretinopathy (PVR), retinal arterial occlusive disease, retinaldetachment, uveitic retinal disease; sympathetic opthalmia; VogtKoyanagi-Harada (VKH) syndrome; uveal diffusion; a posterior ocularcondition caused by or influenced by an ocular laser treatment;posterior ocular conditions caused by or influenced by a photodynamictherapy, photocoagulation, radiation retinopathy, epiretinal membranedisorders, branch retinal vein occlusion, anterior ischemic opticneuropathy, non-retinopathy diabetic retinal dysfunction, retinitispigmentosa, and glaucoma. Glaucoma can be considered a posterior ocularcondition because the therapeutic goal is to prevent the loss of orreduce the occurrence of loss of vision due to damage to or loss ofretinal cells or optic nerve cells (i.e. neuroprotection).

The term “biodegradable polymer” refers to a polymer or polymers whichdegrade in vivo, and wherein erosion of the polymer or polymers overtime occurs concurrent with or subsequent to release of the therapeuticagent. Specifically, hydrogels such as methylcellulose which act torelease drug through polymer swelling are specifically excluded from theterm “biodegradable polymer”. The terms “biodegradable” and“bioerodible” are equivalent and are used interchangeably herein. Abiodegradable polymer may be a homopolymer, a copolymer, or a polymercomprising more than two different polymeric units.

The term “treat”, “treating”, or “treatment” as used herein, refers toreduction or resolution or prevention of an ocular condition, ocularinjury or damage, or to promote healing of injured or damaged oculartissue. A treatment is usually effective to reduce at least one symptomof an ocular condition, ocular injury or damage.

The term “therapeutically effective amount” as used herein, refers tothe level or amount of agent needed to treat an ocular condition, orreduce or prevent ocular injury or damage without causing significantnegative or adverse side effects to the eye or a region of the eye. Inview of the above, a therapeutically effective amount of a therapeuticagent, such as a cyclic lipid, is an amount that is effective inreducing at least one symptom of an ocular condition.

Microspheres have been developed which can release drug loads overvarious time periods. These microspheres, which when inserted into thesubconjunctival space of an eye provide therapeutic levels of a cycliclipid for extended periods of time (e.g., for about 1 week or more). Thedisclosed microspheres are effective in treating ocular conditions, suchas ocular conditions associated with elevated intraocular pressure, andmore specifically in reducing at least one symptom of glaucoma.

Methods for producing microspheres have also been developed. Forexample, the present invention encompasses therapeutic polymericmicroparticles and methods of making and using such microparticles. Asdisclosed herein, the microparticles may be oil-in-oil emulsifiedmicroparticles.

In one embodiment of the present invention, a microsphere comprises abiodegradable polymer matrix. The biodegradable polymer matrix is onetype of a drug release sustaining component. The biodegradable polymermatrix is effective in forming a biodegradable microsphere. Thebiodegradable microsphere comprises a cyclic lipid component associatedwith the biodegradable polymer matrix. The matrix degrades at a rateeffective to sustain release of an amount of the cyclic lipid componentfor a time greater than about one week from the time in which themicrosphere is placed in ocular region or ocular site, such as thesubconjunctival space of an eye.

The cyclic lipid component of the microsphere may include one or moretypes of prostaglandin, prostaglandin analog, prostaglandin derivative,prostamide, prostamide analog, and a prostamide derivative, and anysalts thereof, and mixtures thereof. In certain microspheres, the cycliclipid component may comprise a compound having the following formula(VI)

wherein the dashed bonds represent a single or double bonds which can bein the cis or trans configuration, A is an alkyene or alkenylene radicalhaving from two to six carbon atoms, which radical may be interrupted byone or more oxide radicals and substituted with one or more hydroxy,oxo, alkoxy or alkycarboxyl groups wherein said alkyl radical comprisesfrom one to six carbon atoms; D is a branched or unbranched alkyl orheteroalkyl radical of from two to 10 carbon atoms, a cycloalkyl radicalhaving from three to seven carbon atoms, or an aryl radical, selectedfrom the group consisting of hydrocarbyl aryl and heteroaryl radicalshaving from four to ten carbon atoms wherein the heteroatom is selectedfrom the group consisting of nitrogen, oxygen and sulfur atoms; X is aradical selected from the group consisting of hydrogen, a lower alkylradical having from one to six carbon atoms, R⁵—C(═O)— or R⁵—O—C(═O)—wherein R⁵ is a lower alkyl radical having from one to six carbon atoms;Z is ═O or represents 2 hydrogen radicals; one of R₁ and R² is ═O, —OHor a —O—C(═O)—R⁶ group, and the other one is —OH or —O—C(═O)—R⁶, or R¹is ═O and R² is H, wherein R⁶ is a saturated or unsaturated acyclichydrocarbon group having from 1 to about 20 carbon atoms, or—(CH₂)_(m)R⁷ wherein m is 0-10, and R⁷ is cycloalkyl radical, havingfrom three to seven carbon atoms, or a hydrocarbyl aryl or heteroarylradical, as defined above, or a pharmaceutically acceptable saltthereof.

Pharmaceutically acceptable acid addition salts of certain of the cycliclipids of the invention are those formed from acids which form non-toxicaddition salts containing pharmaceutically acceptable anions, such asthe hydrochloride, hydrobromide, hydroiodide, sulfate, or bisulfate,phosphate or acid phosphate, acetate, maleate, fumarate, oxalate,lactate, tartrate, citrate, gluconate, saccharate and p-toluenesulphonate salts.

In further embodiments, the cyclic lipid component comprises a compoundhaving the following formula (VII)

wherein the radicals are as defined for Formula VI.

In another embodiment, the cyclic lipids of the present invention maycomprise a compound having the following formula (VII)

wherein hatched lines indicate the α configuration and the solidtriangles comprise the β configuration and the radicals are as definedfor Formula VI.

In a further embodiment, the cyclic lipid component comprises a compoundhaving the following formula (VIII)

wherein the radicals are as defined for Formula VI.

In a further embodiment, the cyclic lipid component comprises a compoundhaving the following formula (IX)

wherein X is as defined for Formula VI.

In a further embodiment, the cyclic lipid component comprises a compoundhaving the following formula (X)

In particular, the cyclic lipid component may comprise prostaglandin E1or prostaglandin E2, or salts, esters or mixtures thereof. It will beunderstood that the cyclic lipids used in the present invention include,where appropriate, salts or esters of any compound disclosed herein.

In another embodiment, the cyclic lipid component of the presentinvention may comprise a compound having the formula (I)

wherein the dashed bonds represent a single or double bond which can bein the cis or trans configuration, A is an alkylene or alkenyleneradical having from two to six carbon atoms, which radical may beinterrupted by one or more oxide radicals and substituted with one ormore hydroxy, oxo, alkyloxy or akylcarboxy groups wherein said alkylradical comprises from one to six carbon atoms; B is a cycloalkylradical having from three to seven carbon atoms, or an aryl radical,selected from the group consisting of hydrocarbyl aryl and heteroarylradicals having from four to ten carbon atoms wherein the heteroatom isselected from the group consisting of nitrogen, oxygen and sulfur atoms;X is a radical selected from the group consisting of —OR⁴ and —N(R⁴)₂wherein R⁴ is selected from the group consisting of hydrogen, a loweralkyl radical having from one to six carbon atoms,

wherein R⁵ is a lower alkyl radical having from one to six carbon atoms;Z is ═O or represents 2 hydrogen radicals; one of R₁ and R₂ is ═O, —OHor a —O(CO)R₆ group, and the other one is —OH or —O(CO)R₆, or R₁ is ═Oand R₂ is H, wherein R₆ is a saturated or unsaturated acyclichydrocarbon group having from 1 to about 20 carbon atoms, or —(CH₂)mR₇wherein m is 0 or an integer of from 1 to 10, and R₇ is cycloalkylradical, having from three to seven carbon atoms, or a hydrocarbyl arylor heteroaryl radical, as defined above, or apharmaceutically-acceptable salt thereof, provided, however, that when Bis not substituted with a pendant heteroatom-containing radical, and Zis ═O, then X is not —OR⁴.

Pharmaceutically acceptable acid addition salts of certain of thecompounds of the invention are those formed from acids which formnon-toxic addition salts containing pharmaceutically acceptable anions,such as the hydrochloride, hydrobromide, hydroiodide, sulfate, orbisulfate, phosphate or acid phosphate, acetate, maleate, fumarate,oxalate, lactate, tartrate, citrate, gluconate, saccharate and p-toluenesulphonate salts.

In more specific implants, the compound of the prostamide component hasthe following formula (II)

wherein y is 0 or 1, x is 0 or 1 and x+y are not both 1, Y is a radicalselected from the group consisting of alkyl, halo, nitro, amino, thiol,hydroxy, alkyloxy, alkylcarboxy and halo substituted alkyl, wherein saidalkyl radical comprises from one to six carbon atoms, n is 0 or aninteger of from 1 to 3 and R₃ is ═O, —OH or —O(CO)R₆.

In additional implants, the compound of the prostamide component has thefollowing formula (III)

wherein hatched lines indicate the α configuration and solid trianglesindicate the β configuration.

In certain implants, the compound of the prostamide component has thefollowing formula (IV)

wherein Y¹ is Cl or trifluoromethyl, such as the compound having thefollowing formula (V)

and the 9- and/or 11- and/or 15 esters thereof.

In another embodiment, the compounds used in conjunction with the resentinvention include

-   a) cyclopentane    heptenol-5-cis-2-(3α-hydroxy-5-phenyl-1-trans-pentenyl)-3,5-dihydroxy,    [1α,2β,3α,5α];-   b) cyclopentane    heptenamide-5-cis-2-(3α-hydroxy-5-phenyl-1-trans-pentenyl)-3,5-dihydroxy,    [1α,2β,3α,5α];-   c) cyclopentane    N,N-dimethylheptenamide-5-cis-2-(3α-hydroxy-5-phenyl-1-trans-penten-yl)-3,5-dihydroxy,    [1α,2β,3α,5α];-   d) cyclopentane heptenyl    methoxide-5-cis-2-(3α-hydroxy-5-phenyl-1-trans-pentenyl)-3,5-dihydroxy,    [1α,2β,3α,5α];-   e) cyclopentane heptenyl    ethoxide-5-cis-2-(3α-hydroxy-4-meta-chloro-phenoxy-1-trans-butenyl)-3,5-dihydroxy,    [1α,2β,3α,5α];-   f) cyclopentane    heptenylamide-5-cis-2-(3α-hydroxy-4-meta-chloro-phenoxy-1-trans-butenyl)-3,5-dihydroxy,    [1α,2β,3α,5α];-   g) cyclopentane    heptenylamide-5-cis-2-(3α-hydroxy-4-meta-trifluoromethyl-phenoxy-1-trans-butenyl)-3,5-dihydroxy,    [1α,2β,3α,5α];-   h) cyclopentane N-isopropyl    hepteneamide-5-cis-2-(3α-hydroxy-5-phenyl-1-trans-pentenyl)-3,5-dihydroxy,    [1α,2β,3α,5α];-   i) cyclopentane N-ethyl    heptenamide-5-cis-2-(3α-hydroxy-5-phenyl-1-trans-pentenyl)-3,5-dihydroxy,    [1α,2β,3α,5α];-   j) cyclopentane N-methyl    heptenamide-5-cis-2-(3α-hydroxy-5-phenyl-1-trans-pentenyl)-3,5-dihydroxy,    [1α,2β,3α,5α];-   k) cyclopentane    heptenol-5-cis-2-(3α-hydroxy-4-meta-chlorophenoxy-1-trans-butenyl)-3,5-dihydroxy,    [1α,2β,3α,5α];-   l) cyclopentane    heptenamide-5-cis-2-(3α-hydroxy-4-m-chlorophenoxy-1-trans-butenyl)-3,5-dihydroxy,    [1α,2β,3α,5α], and-   m) cyclopentane    heptenol-5-cis-2-(3α-hydroxy-5-phenylpentyl)3,5-dihydroxy,    [1α,2β,3α,5α].

The prostamide having a name cyclopentane N-ethylheptenamide-5-cis2-cis-2-(3α-hydroxy-5-phenyl-1-trans-pentenyl)-3,5-dihydroxy,[1α,2β,3α,5α], and derivatives, analods, and/or esters thereof, isparticularly preferred in this aspect of the invention. This compound isalso known as bimatoprost and is publicly available in a topicalophthalmic solution under the tradename, Lumigan® (Allergan, Inc., CA).

Thus, the microparticles may comprise a therapeutic component whichcomprises, consists essentially of, or consists of bimatoprost, a saltthereof, or mixtures thereof.

The cyclic lipid component may be in a liquid, derivatized, particulate,or powder form and it may be entrapped by the biodegradable polymermatrix. Usually, cyclic lipid particles will have an effective averagesize less than about 3000 nanometers. In certain implants, the particlesmay have an effective average particle size about an order of magnitudesmaller than 3000 nanometers. For example, the particles may have aneffective average particle size of less than about 500 nanometers. Inadditional implants, the particles may have an effective averageparticle size of less than about 400 nanometers, and in still furtherembodiments, a size less than about 200 nanometers.

The cyclic lipid component of the microspheres is preferably from about10% to 90% by weight of the microspheres. More preferably, the cycliclipid component is from about 20% to about 80% by weight of themicrospheres. In a preferred embodiment, the cyclic lipid componentcomprises about 20% by weight of the microsphere (e.g., 15%-25%). Inanother embodiment, the cyclic lipid component comprises about 50% byweight of the microspheres.

Suitable polymeric materials or compositions for use in the microspheresinclude those materials which are compatible, that is biocompatible,with the eye so as to cause no substantial interference with thefunctioning or physiology of the eye. Such materials preferably are atleast partially and more preferably substantially completelybiodegradable or bioerodible.

Examples of useful polymeric materials include, without limitation, suchmaterials derived from and/or including organic esters and organicethers, which when degraded result in physiologically acceptabledegradation products, including the monomers. Also, polymeric materialsderived from and/or including, anhydrides, amides, orthoesters and thelike, by themselves or in combination with other monomers, may also finduse. The polymeric materials may be addition or condensation polymers,advantageously condensation polymers. The polymeric materials may becross-linked or non-cross-linked, for example not more than lightlycross-linked, such as less than about 5%, or less than about 1% of thepolymeric material being cross-linked. For the most part, besides carbonand hydrogen, the polymers will include at least one of oxygen andnitrogen, advantageously oxygen. The oxygen may be present as oxy, e.g.hydroxy or ether, carbonyl, e.g. non-oxo-carbonyl, such as carboxylicacid ester, and the like. The nitrogen may be present as amide, cyanoand amino. The polymers set forth in Heller, Biodegradable Polymers inControlled Drug Delivery, In: CRC Critical Reviews in Therapeutic DrugCarrier Systems, Vol. 1, CRC Press, Boca Raton, Fla. 1987, pp 39-90,which describes encapsulation for controlled drug delivery, may find usein the present microspheres.

Of additional interest are polymers of hydroxyaliphatic carboxylicacids, either homopolymers or copolymers, and polysaccharides.Polyesters of interest include polymers of D-lactic acid, L-lactic acid,racemic lactic acid, glycolic acid, polycaprolactone, and combinationsthereof. Generally, by employing the L-lactate or D-lactate, a slowlyeroding polymer or polymeric material is achieved, while erosion issubstantially enhanced with the lactate racemate.

Among the useful polysaccharides are, without limitation, calciumalginate, and functionalized celluloses, particularlycarboxymethylcellulose esters characterized by being water insoluble, amolecular weight of about 5 kD to 500 kD, for example.

Other polymers of interest include, without limitation, polyvinylalcohol, polyesters, polyethers and combinations thereof which arebiocompatible and may be biodegradable and/or bioerodible.

Some preferred characteristics of the polymers or polymeric materialsfor use in the present invention may include biocompatibility,compatibility with the therapeutic component, ease of use of the polymerin making the drug delivery systems of the present invention, ahalf-life in the physiological environment of at least about 6 hours,preferably greater than about one day, and water insolubility.

The biodegradable polymeric materials which are included to form thematrix are desirably subject to enzymatic or hydrolytic instability.Water soluble polymers may be cross-linked with hydrolytic orbiodegradable unstable cross-links to provide useful water insolublepolymers. The degree of stability can be varied widely, depending uponthe choice of monomer, whether a homopolymer or copolymer is employed,employing mixtures of polymers, and whether the polymer includesterminal acid groups.

Equally important to controlling the biodegradation of the polymer andhence the extended release profile of the implant is the relativeaverage molecular weight of the polymeric composition employed in themicrospheres. Different molecular weights of the same or differentpolymeric compositions may be included in the microspheres to modulatethe release profile. In certain implants, the relative average molecularweight of the polymer will range from about 9 to about 64 kD, usuallyfrom about 10 to about 54 kD, and more usually from about 12 to about 45kD.

In some microspheres, copolymers of glycolic acid and lactic acid areused, where the rate of biodegradation is controlled by the ratio ofglycolic acid to lactic acid. The most rapidly degraded copolymer hasroughly equal amounts of glycolic acid and lactic acid. Homopolymers, orcopolymers having ratios other than equal, are more resistant todegradation. The ratio of glycolic acid to lactic acid will also affectthe brittleness of the microspheres. The percentage of polylactic acidin the polylactic acid polyglycolic acid (PLGA) copolymer can be 0-100%,preferably about 15-85%, more preferably about 35-65%. In some implants,a 50/50 PLGA copolymer is used.

The biodegradable polymer matrix of the subconjunctival microspheres maycomprise a mixture of two or more biodegradable polymers. For example,the microspheres may comprise a mixture of a first biodegradable polymerand a different second biodegradable polymer. One or more of thebiodegradable polymers may have terminal acid groups.

Release of a drug from an erodible polymer is the consequence of severalmechanisms or combinations of mechanisms. Some of these mechanismsinclude desorption from the microsphere's surface, dissolution,diffusion through porous channels of the hydrated polymer and erosion.Erosion can be bulk or surface or a combination of both. As discussedherein, the matrix of the microspheres may release drug at a rateeffective to sustain release of an amount of the prostamide componentfor more than one week after implantation into an eye. In certainmicrospheres, therapeutic amounts of the cyclic lipid component arereleased for no more than about 30-35 days after administration to thesubconjunctival space. For example, a microsphere may comprisebimatoprost, and the matrix of the microsphere degrades at a rateeffective to sustain release of a therapeutically effective amount ofbimatoprost for about one month after being placed under theconjunctiva. As another example, the microspheres may comprisebimatoprost, and the matrix releases drug at a rate effective to sustainrelease of a therapeutically effective amount of bimatoprost for morethan forty days, such as for about six months.

One example of the biodegradable microsphere comprises an cyclic lipidcomponent associated with a biodegradable polymer matrix, whichcomprises a mixture of different biodegradable polymers. At least one ofthe biodegradable polymers is a polylactide having a molecular weight ofabout 63.3 kD. A second biodegradable polymer is a polylactide having amolecular weight of about 14 kD. Such a mixture is effective insustaining release of a therapeutically effective amount of the cycliclipid component for a time period greater than about one month from thetime the microspheres are placed administered under the conjuctiva.

Another example of a biodegradable microsphere comprises a cyclic lipidcomponent associated with a biodegradable polymer matrix, whichcomprises a mixture of different biodegradable polymers, eachbiodegradable polymer having an inherent viscosity from about 0.16 dl/gto about 1.0 dl/g. For example, one of the biodegradable polymers mayhave an inherent viscosity of about 0.3 dl/g. A second biodegradablepolymer may have an inherent viscosity of about 1.0 dl/g. Additionalmicrospheres may comprise biodegradable polymers that have an inherentviscosity between about 0.2 dl/g and 0.5 dl/g. The inherent viscositiesidentified above may be determined in 0.1% chloroform at 25° C.

One particular microsphere formulation comprises bimatoprost associatedwith a combination of two different polylactide polymers. Thebimatoprost is present in about 20% by weight of the microsphere. Onepolylactide polymer has a molecular weight of about 14 kD and aninherent viscosity of about 0.3 dl/g, and the other polylactide polymerhas a molecular weight of about 63.3 kD and an inherent viscosity ofabout 1.0 dl/g. The two polylactide polymers are present in themicrosphere in a 1:1 ratio. Such a microsphere may be effective inreleasing the bimatoprost for more than two months.

The release of the cyclic lipid component from microspheres into thesubconjuctiva may include an initial burst of release followed by agradual increase in the amount of the cyclic lipid component released,or the release may include an initial delay in release of the prostamidecomponent followed by an increase in release. When the microspheres aresubstantially completely degraded, the percent of the cyclic lipidcomponent that has been released is about one hundred. The microspheredisclosed herein do not completely release, or release about 100% of thecyclic lipid component, until after about one week of being placed in aneye.

It may be desirable to provide a relatively constant rate of release ofthe cyclic lipid component from the microspheres over the life of theimplant. For example, it may be desirable for the cyclic lipid componentto be released in amounts from about 0.01 μg to about 2 μg per day forthe life of the microspheres. However, the release rate may change toeither increase or decrease depending on the formulation of thebiodegradable polymer matrix. In addition, the release profile of theprostamide component may include one or more linear portions and/or oneor more non-linear portions. Preferably, the release rate is greaterthan zero once the microspheres has begun to degrade or erode.

The microspheres may be monolithic, i.e. having the active agent oragents homogenously distributed through the polymeric matrix, orencapsulated, where a reservoir of active agent is encapsulated by thepolymeric matrix. Due to ease of manufacture, monolithic implants areusually preferred over encapsulated forms. However, the greater controlafforded by the encapsulated microspheres may be of benefit in somecircumstances, where the therapeutic level of the drug falls within anarrow window. In addition, the therapeutic component, including thecyclic lipid component, may be distributed in a non-homogenous patternin the matrix. For example, the microspheres may include a portion thathas a greater concentration of the cyclic lipid component relative to asecond portion of the microspheres.

The microspheres disclosed herein may have a size of between about 5 μmand about 1 mm, or between about 10 μm and about 0.8 mm foradministration with a needle. For needle-injected microspheres, themicrosphere may have any appropriate dimensions so long as the longestdimension of the microsphere permits the microsphere to move through aneedle. This is generally not a problem in the administration ofmicrospheres. The subconjunctival space in humans is able to accommodaterelatively large volumes of microspheres, for example, about 100 μl, orabout 150 μl, or about 50-200 μl or more.

The total weight of microsphere in a single dosage an optimal amount,depending on the volume of the subconjunctival space and the activity orsolubility of the active agent. Most often, the dose is usually about 10mg to about 500 mg of microspheres per dose. For example, a singlesubconjunctival injection may contain about 20 mg, or about 50 mg, orabout 75 mg, or about 100 mg, or about 125 mg or about 150 mg, or about175 mg, or about 200 mg of microspheres, including the incorporatedtherapeutic component. For non-human individuals, the dimensions andtotal weight of the microsphere(s) may be larger or smaller, dependingon the type of individual.

The dosage of the therapeutic component in the microsphere is generallyin the range from about 0.001% to about 100 mg per eye per dose, butalso can vary from this depending upon the activity of the agent and itssolubility.

Thus, microspheres can be prepared where the center may be of onematerial and the surface may have one or more layers of the same or adifferent composition, where the layers may be cross-linked, or of adifferent molecular weight, different density or porosity, or the like.For example, where it is desirable to quickly release an initial bolusof drug, the center of the microsphere may be a polylactate coated witha polylactate-polyglycolate copolymer, so as to enhance the rate ofinitial degradation. Alternatively, the center may be polyvinyl alcoholcoated with polylactate, so that upon degradation of the polylactateexterior the center would dissolve and be rapidly washed out of the eye.

The microspheres may be of any particulate geometry including micro andnanospheres, micro and nanoparticles, spheres, powders, fragments andthe like. The upper limit for the microsphere size will be determined byfactors such as toleration for the implant, size limitations oninsertion, desired rate of release, ease of handling, etc. Spheres maybe in the range of about 0.5 μm to 4 mm in diameter, with comparablevolumes for other shaped particles.

The size and form of the microspheres can also be used to control therate of release, period of treatment, and drug concentration at the siteof implantation. Larger microspheres will deliver a proportionatelylarger dose, but depending on the surface to mass ratio, may have aslower release rate. The particular size and geometry of themicrospheres are chosen to suit the activity of the active agent and thelocation of its target tissue.

The proportions of the cyclic lipid component, polymer, and any othermodifiers may be empirically determined by formulating severalmicrosphere batches with varying average proportions. A USP approvedmethod for dissolution or release test can be used to measure the rateof release (USP 23; NF 18 (1995) pp. 1790-1798). For example, using theinfinite sink method, a weighed sample of the microspheres is added to ameasured volume of a solution containing 0.9% NaCl in water, where thesolution volume will be such that the drug concentration is afterrelease is less than 5% of saturation. The mixture is maintained at 37°C. and stirred slowly to maintain the microspheres in suspension. Theappearance of the dissolved drug as a function of time may be followedby various methods known in the art, such as spectrophotometrically,HPLC, mass spectroscopy, etc. until the absorbance becomes constant oruntil greater than 90% of the drug has been released.

In addition to the cyclic lipid component included in the microspheresdisclosed herein, the microsphere may also include one or moreadditional ophthalmically acceptable therapeutic agents. For example,the microspheres may include one or more antihistamines, one or moreantibiotics, one or more beta blockers, one or more steroids, one ormore antineoplastic agents, one or more immunosuppressive agents, one ormore antiviral agents, one or more antioxidant agents, and mixturesthereof. Alternatively, a single injection of microspheres may includetwo or more microsphere batches each containing a different therapeuticcomponent or components. Such a mixture of different microspheres inincluded within the present invention so long as a therapeutic componentcomprises a cyclic lipid.

Additional pharmacologic or therapeutic agents which may find use in thepresent systems, include, without limitation, those disclosed in U.S.Pat. No. 4,474,451, columns 4-6 and U.S. Pat. No. 4,327,725, columns7-8.

Examples of antihistamines include, and are not limited to, loradatine,hydroxyzine, diphenhydramine, chlorpheniramine, brompheniramine,cyproheptadine, terfenadine, clemastine, triprolidine, carbinoxamine,diphenylpyraline, phenindamine, azatadine, tripelennamine,dexchlorpheniramine, dexbrompheniramine, methdilazine, and trimprazinedoxylamine, pheniramine, pyrilamine, chiorcyclizine, thonzylamine, andderivatives thereof.

Examples of antibiotics include without limitation, cefazolin,cephradine, cefaclor, cephapirin, ceftizoxime, cefoperazone, cefotetan,cefutoxime, cefotaxime, cefadroxil, ceftazidime, cephalexin,cephalothin, cefamandole, cefoxitin, cefonicid, ceforanide, ceftriaxone,cefadroxil, cephradine, cefuroxime, ampicillin, amoxicillin,cyclacillin, ampicillin, penicillin G, penicillin V potassium,piperacillin, oxacillin, bacampicillin, cloxacillin, ticarcillin,azlocillin, carbenicillin, methicillin, nafcillin, erythromycin,tetracycline, doxycycline, minocycline, aztreonam, chloramphenicol,ciprofloxacin hydrochloride, clindamycin, metronidazole, gentamicin,lincomycin, tobramycin, vancomycin, polymyxin B sulfate, colistimethate,colistin, azithromycin, augmentin, sulfamethoxazole, trimethoprim, andderivatives thereof.

Examples of beta blockers include acebutolol, atenolol, labetalol,metoprolol, propranolol, timolol, and derivatives thereof.

Examples of steroids include corticosteroids, such as cortisone,prednisolone, flurometholone, dexamethasone, medrysone, loteprednol,fluazacort, hydrocortisone, prednisone, betamethasone, prednisone,methylprednisolone, riamcinolone hexacatonide, paramethasone acetate,diflorasone, fluocinonide, fluocinolone, triamcinolone, derivativesthereof, and mixtures thereof.

Examples of antineoplastic agents include adriamycin, cyclophosphamide,actinomycin, bleomycin, duanorubicin, doxorubicin, epirubicin,mitomycin, methotrexate, fluorouracil, carboplatin, carmustine (BCNU),methyl-CCNU, cisplatin, etoposide, interferons, camptothecin andderivatives thereof, phenesterine, taxol and derivatives thereof,taxotere and derivatives thereof, vinblastine, vincristine, tamoxifen,etoposide, piposulfan, cyclophosphamide, and flutamide, and derivativesthereof.

Examples of immunosuppressive agents include cyclosporine, azathioprine,tacrolimus, and derivatives thereof.

Examples of antiviral agents include interferon gamma, zidovudine,amantadine hydrochloride, ribavirin, acyclovir, valciclovir,dideoxycytidine, phosphonoformic acid, ganciclovir, and derivativesthereof.

Examples of antioxidant agents include ascorbate, alpha-tocopherol,mannitol, reduced glutathione, various carotenoids, cysteine, uric acid,taurine, tyrosine, superoxide dismutase, lutein, zeaxanthin,cryotpxanthin, astazanthin, lycopene, N-acetyl-cysteine, carnosine,gamma-glutamylcysteine, quercitin, lactoferrin, dihydrolipoic acid,citrate, Ginkgo Biloba extract, tea catechins, bilberry extract,vitamins E or esters of vitamin E, retinyl palmitate, and derivativesthereof.

Other therapeutic agents include squalamine, carbonic anhydraseinhibitors, alpha-2 adrenergic receptor agonists, antiparasitics,antifungals, and derivatives thereof.

The amount of active agent or agents employed in the microspheres,individually or in combination, will vary widely depending on theeffective dosage required and the desired rate of release from themicrospheres. Usually the agent will be at least about 1, more usuallyat least about 10 weight percent of the microsphere, and usually notmore than about 80, more usually not more than about 40 weight percentof the microspheres.

Some of the present implants may comprise a cyclic lipid component thatcomprises a combination of two or more different cyclic lipidderivatives. One microsphere or dosage of microspheres may comprise acombination of bimatoprost and latanoprost. Another microsphere ordosage of microspheres may comprise a combination of bimatoprost andtravoprost.

As discussed herein, the present microspheres may comprise additionaltherapeutic agents. For example, one microsphere or dosage ofmicrospheres may comprise a combination of bimatoprost and abeta-adrenergic receptor antagonist. More specifically, the microsphereor dosage of microspheres may comprise a combination of bimatoprost andTimolol®. Or, a microsphere or dosage of microspheres may comprise acombination of bimatoprost and a carbonic anyhdrase inhibitor. Forexample, the microsphere or dosage of microspheres may comprise acombination of bimatoprost and dorzolamide (Trusopt®).

In addition to the therapeutic component, the microspheres disclosedherein may include or may be provided in compositions that includeeffective amounts of buffering agents, preservatives and the like.Suitable water soluble buffering agents include, without limitation,alkali and alkaline earth carbonates, phosphates, bicarbonates,citrates, borates, acetates, succinates and the like, such as sodiumphosphate, citrate, borate, acetate, bicarbonate, carbonate and thelike. These agents advantageously present in amounts sufficient tomaintain a pH of the system of between about 2 to about 9 and morepreferably about 4 to about 8. As such the buffering agent may be asmuch as about 5% by weight of the total implant. Suitable water solublepreservatives include sodium bisulfite, sodium bisulfate, sodiumthiosulfate, ascorbate, benzalkonium chloride, chlorobutanol,thimerosal, phenylmercuric acetate, phenylmercuric borate,phenylmercuric nitrate, parabens, methylparaben, polyvinyl alcohol,benzyl alcohol, phenylethanol and the like and mixtures thereof. Theseagents may be present in amounts of from about 0.001% to about 5% byweight and preferably about 0.01% to about 2% by weight. In at least oneof the present microspheres, a benzylalkonium chloride preservative isprovided in the implant, such as when the cyclic lipid componentconsists essentially of bimatoprost.

In some situations mixtures of microspheres may be utilized employingthe same or different pharmacological agents. In this way, a cocktail ofrelease profiles, giving a biphasic or triphasic release with a singleadministration is achieved, where the pattern of release may be greatlyvaried.

Additionally, release modulators such as those described in U.S. Pat.No. 5,869,079 may be included in the microspheres. The amount of releasemodulator employed will be dependent on the desired release profile, theactivity of the modulator, and on the release profile of the cycliclipid component in the absence of modulator. Electrolytes such as sodiumchloride and potassium chloride may also be included in themicrospheres. Where the buffering agent or enhancer is hydrophilic, itmay also act as a release accelerator. Hydrophilic additives act toincrease the release rates through faster dissolution of the materialsurrounding the drug in the microspheres, which increases the surfacearea of the drug exposed, thereby increasing the rate of drugbioerosion. Similarly, a hydrophobic buffering agent or enhancerdissolves more slowly, slowing the exposure of drug, and thereby slowingthe rate of drug bioerosion.

In certain microspheres, the combination of bimatoprost and abiodegradable polymer matrix is released or delivered an amount ofbimatoprost between about 0.1 mg to about 0.5 mg for about 3-6 monthsafter implantation into the eye.

Various techniques may be employed to produce the microspheres describedherein. Useful techniques include, but are not necessarily limited to,self-emulsification methods, super critical fluid methods, solventevaporation methods, phase separation methods, spray drying methods,grinding methods, interfacial methods, molding methods, injectionmolding methods, combinations thereof and the like.

Compression methods may be used to make the implants, and typicallyyield implants with faster release rates than extrusion methods.Compression methods may use pressures of about 50-150 psi, morepreferably about 70-80 psi, even more preferably about 76 psi, and usetemperatures of about 0 degrees C. to about 115 degrees C., morepreferably about 25 degrees C.

Another method comprises using an oil-in-oil emulsion process to producethe present microparticles.

In one embodiment, a method for producing therapeutic polymericmicroparticles comprises encapsulating a cyclic lipid component with apolymeric component to form a population of cyclic lipid-encapsulatedmicroparticles by an oil-in-oil emulsion process. Such microparticlesare effective in treating one or more ocular conditions, as describedherein, and are suitable for administration to a patient into thesubconjunctival space. The therapeutic activity of the cyclic lipidcomponent remains stable during storage of the microspheres which may beattributed to the particular encapsulated form of the microspheres.

In more detail, a method of forming microparticles comprises forming anoil-in-oil emulsion containing the cyclic lipid component and thepolymeric component. For example, the method may include mixing orcombining a plurality, such as two or more, non-aqueous liquidcompositions to form an emulsion. In one embodiment, a first compositionmay comprise an organic solvent, and a second composition may comprisean oil. At least one of the compositions contains the cyclic lipidcomponent, the polymeric component, or the cyclic lipid component andthe polymeric component.

The method also comprises drying the emulsion to form a dried emulsionproduct. The drying can be achieved using one or more techniques toremove the liquid from the emulsified product. For example, the dryingprocess can include increasing the temperature of or near the emulsifiedproduct to facilitate evaporation of the liquid, can include the use ofa vacuum to facilitate removal of the liquid, can include centrifugationto separate solid from the liquid, and combinations thereof.

The dried emulsion product can then be contacted with a solvent to forma solvent containing composition. Adding a solvent to the dried emulsionproduct, or otherwise contacting the product with the solvent results inthe product being suspended in a volume of the liquid solvent. Thecontacting can include a step of stirring or mixing the combination ofthe solvent and dried emulsion product to form a suspension of the driedemulsion product in the solvent. The suspension can include particles ofthe dried emulsion product, for example, microspheres of various sizesand shapes, including microparticles.

After forming the solvent containing composition, the method cancomprise removing the solvent from the solvent-containing composition toform a population of microparticles that comprise the cyclic lipidcomponent and the polymeric component. The removing can include one ormore steps of centrifuging and/or rinsing intermediate compositions, andcan include one or more steps of drying the resulting composition. Theresulting products are encapsulated microparticles or microimplants thatcomprise a cyclic lipid component encapsulated by a polymeric component,such as a biodegradable polymer coating.

As discussed herein, the cyclic lipid component can comprises a singletype of cyclic lipid derivative or derivatives. In certain embodiments,the cyclic lipid component comprises at least one prostamide derivativeselected from the group consisting of bimatoprost, esters thereof, andmixtures thereof. In a further embodiment, the cyclic lipid componentconsists essentially of bimatoprost.

In additional embodiments, the cyclic lipid component can comprisecombinations of two or more different cyclic lipid derivatives, such asa combination of bimatoprost and iatanoprost, bimatoprost andtravoprost, and the like.

The present methods are effective in producing encapsulated cyclic lipidcomponent microparticles that maintain or preserve a substantialportion, if not all, of the therapeutic activity after a terminalsterilization procedure. It can be understood, that the present methodsmay also comprise a step of terminally sterilizing the microparticles.The microparticles can be sterilized before packaging or in theirpackaging. Sterilization of packages containing the presentmicroparticles or implants is often preferred. The method may compriseexposing the present microparticles or implants to sterilizing amountsof gamma radiation, e-beam radiation, and other terminal sterilizationproducts. In one embodiment, a method may comprise a step of exposingthe present microparticles to gamma radiation at a dose of about 25 kGy.

As discussed herein, the polymeric component recited in the presentmethod may comprise a biodegradable polymer or biodegradable copolymer.In at least one embodiment, the polymeric component comprises a poly(lactide-co-glycolide) PLGA copolymer. In a further embodiment, the PLGAcopolymer has a lactide/glycolide ratio of 75/25. In a still furtherembodiment, the PLGA copolymer has at least one of a molecular weight ofabout 63 kilodaltons and an inherent viscosity of about 0.6 dL/g.

The present methods may also comprise a step of forming a firstcomposition which comprises a cyclic lipid component, a polymericcomponent, and an organic solvent, and a step of forming a secondoil-containing composition, and mixing the first composition and thesecond oil-containing composition.

The present methods may also comprise evaporating the oil-in-oilemulsion to form an evaporated product, as described herein.

Further, the methods may comprise a step of suspending the evaporatedproduct in a solvent before removing the solvent from the solventcontaining composition. Such a step can be understood to be a way offorming a suspension.

As one example, a method of forming encapsulated bimatoprostbiodegradable microparticles comprises forming an oil-in-oil emulsioncomprising the bimatoprost and PLGA, evaporating the liquid from theemulsion to form an evaporated product, suspending and rinsing theevaporated product, and drying the evaporated product.

In accordance with the disclosure herein, an embodiment of the presentinvention is a population of microparticles that comprise a polymericcomponent encapsulating a prostamide component in the form of oil-in-oilemulsified microparticles. In one specific embodiment, the polymericcomponent comprises a PLGA copolymer and the cyclic lipid componentcomprises at least one prostamide derivative selected from the groupconsisting of bimatoprost, salts thereof, and mixtures thereof.

The resulting population may be a terminally sterilized population ofmicroparticles. Terminally sterilized microparticles retain theirtherapeutic activity during storage and therefore can provide successfultreatment to patients. In certain embodiments, a major portion of theprostamide component of the terminally sterilized microparticles remainsstable. For example, in certain embodiments, at least 80% of theprostamide component remains stable after sterilization. In furtherembodiments, at least 90%, at least 95%, or at least 99% of theprostamide component remains stable.

In addition, the present population of microparticles may have a maximumparticle diameter less than about 200 μm. In certain embodiments, thepopulation of microparticles has an average or mean particle diameterless than about 50 μm. In further embodiments, the population ofmicroparticles has a mean particle diameter from about 30 μm to about 50μm.

The present microparticles are structured or configured to release thecyclic lipid component for extended periods of time at controlled rates.In some embodiments, the cyclic lipid component is released at asubstantially linear rate (e.g., a single rate) over the life of themicroparticles (e.g., until the microparticles fully degrade). Otherembodiments are capable of releasing the cyclic lipid component atmultiple rates or different rates over the life of the microparticles.The rate at which the microparticles degrade can vary, as discussedherein, and therefore, the present microparticles can release the cycliclipid component for different periods of time depending on theparticular configuration and materials of the microparticles. In atleast one embodiment, a microparticle can release about 1% of the cycliclipid component in the microparticles per day. In a further embodiment,the microparticles may have a release rate of about 0.7% per day whenmeasured in vitro. Thus, over a period of about 40 days, about 30% ofthe cyclic lipid component may have been released.

As discussed herein, the amount of the cyclic lipid component present inthe microparticles can vary. In certain embodiments, about 10% wt/wt ofthe microparticles is the cyclic lipid component. In furtherembodiments, the cyclic lipid component constitutes about 5% wt/wt ofthe microparticles.

The microspheres, including the population of microparticles, of thepresent invention may be inserted into the subconjunctival space of aneye by a variety of methods. The method of placement may influence thetherapeutic component or drug release kinetics. A preferred means ofadministration of the microspheres of the present invention is bysubconjunctival injection. The location of the site of injection of themicrospheres may influence the concentration gradients of therapeuticcomponent or drug surrounding the element, and thus influence thedelivery rate to a given tissue of the eye. For example, an injectioninto the conjunctiva toward the posterior of the eye will direct drugmore efficiently to the tissues of the posterior segment, while a siteof injection closer to the anterior of the eye (but avoiding the cornea)may direct drug more efficiently to the anterior segment.

Microparticles may be administered to patients by administering anophthalmically acceptable composition which comprises the microparticlesto the patient. For example, microparticles may be provided in a liquidcomposition, a suspension, an emulsion, and the like, and administeredby injection or implantation into the subconjunctival space of the eye.

The present implants or microparticles are configured to release anamount of cyclic lipid component effective to treat an ocular condition,such as by reducing at least one symptom of the ocular condition. Morespecifically, the microparticles may be used in a method to treatglaucoma, such as open angle glaucoma, ocular hypertension, chronicangle-closure glaucoma, with patent iridotomy, psuedoexfoliativeglaucoma, and pigmentary glaucoma. By injecting the cyclic lipidcomponent-containing microspheres into the subconjunctival space of aneye, it is believed that the cyclic lipid component is effective toenhance aqueous humor flow thereby reducing intraocular pressure.Additionally, the present inventors have shown that subconjunctivaldelivery of microspheres containing a cyclic lipid component is able toprovide quite high concentrations of the therapeutic agent to the retinaof the eye.

The microspheres disclosed herein may also be configured to release thecyclic lipid component with or without additional agents, as describedabove, which to prevent or treat diseases or conditions, such as thefollowing:

MACULOPATHIES/RETINAL DEGENERATION: Non-Exudative Age Related MacularDegeneration (ARMD), Exudative Age Related Macular Degeneration (ARMD),Choroidal Neovascularization, Diabetic Retinopathy, Acute MacularNeuroretinopathy, Central Serous Chorioretinopathy, Cystoid MacularEdema, Diabetic Macular Edema.

UVEITIS/RETINITIS/CHOROIDITIS: Acute Multifocal Placoid PigmentEpitheliopathy, Behcet's Disease, Birdshot Retinochoroidopathy,Infectious (Syphilis, Lyme, Tuberculosis, Toxoplasmosis), IntermediateUveitis (Pars Planitis), Multifocal Choroiditis, Multiple EvanescentWhite Dot Syndrome (MEWDS), Ocular Sarcoidosis, Posterior Scleritis,Serpignous Choroiditis, Subretinal Fibrosis and Uveitis Syndrome,Vogt-Koyanagi-Harada Syndrome.

VASCULAR DISEASES/EXUDATIVE DISEASES: Coat's Disease, ParafovealTelangiectasis, Papillophlebitis, Frosted Branch Angitis, Sickle CellRetinopathy and other Hemoglobinopathies, Angioid Streaks, FamilialExudative Vitreoretinopathy.

TRAUMATIC/SURGICAL: Sympathetic Ophthalmia, Uveitic Retinal Disease,Retinal Detachment, Trauma, Laser, PDT, Photocoagulation, HypoperfusionDuring Surgery, Radiation Retinopathy, Bone Marrow TransplantRetinopathy.

PROLIFERATIVE DISORDERS: Proliferative Vitreal Retinopathy andEpiretinal Membranes, Proliferative Diabetic Retinopathy.

INFECTIOUS DISORDERS: Ocular Histoplasmosis, Ocular Toxocariasis,Presumed Ocular Histoplasmosis Syndrome (POHS), Endophthalmitis,Toxoplasmosis, Retinal Diseases Associated with HIV Infection, ChoroidalDisease Associated with HIV Infection, Uveitic Disease Associated withHIV Infection, Viral Retinitis, Acute Retinal Necrosis, ProgressiveOuter Retinal Necrosis, Fungal Retinal Diseases, Ocular Syphilis, OcularTuberculosis, Diffuse Unilateral Subacute Neuroretinitis, Myiasis.

GENETIC DISORDERS: Systemic Disorders with Associated RetinalDystrophies, Congenital Stationary Night Blindness, Cone Dystrophies,Fundus Flavimaculatus, Best's Disease, Pattern Dystrophy of the RetinalPigmented Epithelium, X-Linked Retinoschisis, Sorsby's Fundus Dystrophy,Benign Concentric Maculopathy, Bietti's Crystalline Dystrophy,pseudoxanthoma elasticum.

RETINAL TEARS/HOLES: Retinal Detachment, Macular Hole, Giant RetinalTear.

TUMORS: Retinal Disease Associated with Tumors, Congenital Hypertrophyof the RPE, Posterior Uveal Melanoma, Choroidal Hemangioma, ChoroidalOsteoma, Choroidal Metastasis, Combined Hamartoma of the Retina andRetinal Pigmented Epithelium, Retinoblastoma, Vasoproliferative Tumorsof the Ocular Fundus, Retinal Astrocytoma, Intraocular Lymphoid Tumors.

MISCELLANEOUS: Punctate Inner Choroidopathy, Acute Posterior MultifocalPlacoid Pigment Epitheliopathy, Myopic Retinal Degeneration, AcuteRetinal Pigment Epithelitis and the like.

In one embodiment, an implant, such as the microspheres disclosedherein, is administered to a subconjunctival space of an eye.

In at least one embodiment, a method of reducing intraocular pressure inan eye of a patient comprises administering a microsphere containing acyclic lipid component, as disclosed herein, to a patient bysubconjuctival injection. A syringe apparatus including an appropriatelysized needle, for example, a 22 gauge needle, a 27 gauge needle or a 30gauge needle, can be effectively used to inject the composition withinto the subconjunctival space of an eye of a human or animal. Frequentrepeat injections are often not necessary due to the extended release ofthe cyclic lipid component from the microspheres.

In addition, for dual therapy approaches to treating an ocularcondition, the method may include one or more additional steps ofadministering additional therapeutic agents to the eye, such as bytopically administering compositions containing timolol, dorzolamide,and iatoprost, among others.

In another aspect of the invention, kits and packages for treating anocular condition of the eye are provided, comprising: a) a containercomprising an extended release microsphere formulation comprising atherapeutic component including a cyclic lipid component, such asbimatoprost (Lumigan®), and a drug release sustaining component; and b)instructions for use. Instructions may include steps of how to handlethe microspheres, how to insert the microspheres into an ocular region,and what to expect from using the microspheres.

In certain implants, the microspheres preparation comprises atherapeutic component which consists essentially of bimatoprost, saltsthereof, and mixtures thereof, and a biodegradable polymer matrix. Thebiodegradable polymer matrix may consist essentially of PLA, PLGA, or acombination thereof. When placed in the eye, the preparation releasesabout 40% to about 60% of the bimatoprost to provide a loading dose ofthe bimatoprost within about one day after subconjunctivaladministration. Subsequently, the microspheres release about 1% to about2% of the bimatoprost per day to provide a sustained therapeutic effect.Such microsphere preparations may be effective in reducing andmaintaining a reduced intraocular pressure, such as below about 15 mm Hgfor several months, and potentially for one or two years.

Other microspheres disclosed herein may be configured such that theamount of the cyclic lipid component that is released from themicrospheres within two days of subconjunctival injection is less thanabout 95% of the total amount of the cyclic lipid component in themicrosphere. In certain formulations, 95% of the cyclic lipid componentis not released until after about one week of injection. In certainmicrosphere formulations, about 50% of the cyclic lipid component isreleased within about one day of placement in the eye, and about 2% isreleased for about 1 month after being placed in the eye. In othermicrospheres, about 50% of the cyclic lipid component is released withinabout one day of subconjunctival administration, and about 1% isreleased for about 2 months after such administration.

In an alternative embodiment, the present invention may comprise theinstallation of a hollow depot device, made from a biocompatible,preferably non-biodegradable, polymer in connection with and preferablypenetrating the conjunctiva of an eye. Such a device is described, forexample, in US Patent Publication 20050112175, hereby incorporated byreference herein. The depot is preferably refillable with microspherescomprising an ophthalmically active therapeutic component, such as anophthalmically active cyclic lipid component.

EXAMPLES Example 1 Manufacture and Testing of Implants ContainingBimatoprost and a Biodegradable Polymer Matrix

Biodegradable implants were made by combining bimatoprost with abiodegradable polymer composition. 800 mg of polylactic acid (PLA) wascombined with 200 mg of bimatoprost. The combination was dissolved in 25milliliters of dichloromethane. The mixture was placed in a vacuum at45° C. overnight to evaporate the dichloromethane. The resulting mixturewas in the form of a cast sheet. The cast sheet was cut and ground in ahigh shear grinder with dry ice until the particles could pass through asieve having a pore size of about 125 μm. The percent of bimatoprostpresent in the microparticles was analyzed using high pressure liquidchromatography (HPLC). The percent release of bimatoprost from themicroparticles was profiled using dialysis. The percent of bimatoprostremaining in the recovered particles was analyzed by HPLC.

The release profile is described in Table 1.

Time Elapsed Time Percent Percent Per Point (Days) Released Day Start 0— — 1 1.03 47.51 47.51  2 2.03 47.92 0.41 3 3.03 49.99 2.07 4 4.03 50.090.10 5 7.04 50.90 0.82

The percent loading of bimatoprost was 14.93%. The percent ofbimatoprost remaining in the recovered release particles was 4.94%.

Example 2 Extrusion Process and Compression of ManufacturingBimatoprost-Containing Biodegradable Intraocular Implants

Bimatoprost is combined with a biodegradable polymer composition in amortar. The combination is mixed with a shaker set at about 96 RPM forabout 15 minutes. The powder blend is scraped off the wall of the mortarand is then remixed for an additional 15 minutes. The mixed powder blendis heated to a semi-molten state at specified temperature for a total of30 minutes, forming a polymer/drug melt.

Rods are manufactured by pelletizing the polymer/drug melt using a 9gauge polytetrafluoroethylene (PTFE) tubing, loading the pellet into thebarrel and extruding the material at the specified core extrusiontemperature into filaments. The filaments are then cut into about 1 mgsize implants or drug delivery systems. The rods may have dimensions ofabout 2 mm long×0.72 mm diameter. The rod implants weigh between about900 μg and 1100 μg.

Wafers are formed by flattening the polymer melt with a Carver press ata specified temperature and cutting the flattened material into wafers,each weighing about 1 mg. The wafers have a diameter of about 2.5 mm anda thickness of about 0.13 mm. The wafer implants weigh between about 900μg and 1100 μg.

In-vitro release testing is performed by placing each implant into a 24mL screw cap vial with 10 mL of Phosphate Buffered Saline solution at37° C. 1 mL aliquots are removed and are replaced with equal volume offresh medium on day 1, 4, 7, 14, 28, and every two weeks thereafter.

Drug assays are performed by HPLC, which consists of a Waters 2690Separation Module (or 2696), and a Waters 2996 Photodiode ArrayDetector. An Ultrasphere, C-18 (2), 5 m; 4.6×150 mm column heated at 30°C. is used for separation and the detector is set at about 264 nm. Themobile phase is (10:90) MeOH-buffered mobile phase with a flow rate of 1mL/min and a total run time of 12 min per sample. The buffered mobilephase may comprise (68:0.75:0.25:31) 13 mM 1-Heptane Sulfonic Acid,sodium salt-glacial acetic acid-triethylamine-Methanol. The releaserates are determined by calculating the amount of drug being released ina given volume of medium over time in g/day.

Polymers which may be used in the implants can be obtained fromBoehringer Ingelheim. Examples of polymer include: RG502, RG752, R202H,R203 and R206, and Purac PDLG (50/50). RG502 is (50:50)poly(D,L-lactide-co-glycolide), RG752 is (75:25)poly(D,L-lactide-co-glycolide), R202H is 100% poly(D, L-lactide) withacid end group or terminal acid groups, R203 and R206 are both 100%poly(D, L-lactide). Purac PDLG (50/50) is (50:50)poly(D,L-lactide-co-glycolide). The inherent viscosity of RG502, RG752,R202H, R203, R206, and Purac PDLG are 0.2, 0.2, 0.2, 0.3, 1.0, and 0.2dL/g, respectively. The average molecular weight of RG502, RG752, R202H,R203, R206, and Purac PDLG are, 11700, 11200, 6500, 14000, 63300, and9700 daltons, respectively.

Example 3 Bimatoprost/PLA/PLGA Intraocular Implants to Treat Glaucoma

A 72 year old female suffering from glaucoma in both eyes receives anintraocular implant containing bimatoprost and a combination of a PLAand PLGA in each eye. The implants weigh about 1 mg, and contain about500 mg of bimatoprost. One implant is placed in the vitreous of each eyeusing a syringe. In about two days, the patient reports a substantialrelief in ocular comfort. Examination reveals that the intraocularpressure has decreased, the average intraocular pressure measured at8:00 AM has decreased from 28 mm Hg to 14.3 mm Hg. The patient ismonitored monthly for about 6 months. Intraocular pressure levels remainbelow 15 mm Hg for six months, and the patient reports reduced oculardiscomfort.

Example 4 Bimatoprost/PLA Intraocular Implants to Reduce OcularHypertension

A 62 year old male presents with an intraocular pressure in his left eyeof 33 mm Hg. An implant containing 400 mg of bimatoprost and 600 mg ofPLA is inserted into the vitreous of the left eye using a trocar. Thepatient's intraocular pressure is monitored daily for one week, and thenmonthly thereafter. One day after implantation, the intraocular pressureis reduced to 18 mm Hg. By day 7 after implantation, the intraocularpressure is relatively stable at 14 mm Hg. The patient does notexperience any further signs of elevated intraocular pressure for 2years.

Example 5 Oil in Oil Method for Microencapsulation of a ProstamideDerivative

This example describes a process for producing microparticles thatinclude a prostamide derivative encapsulated by a biodegradable polymer.In the specific example, bimatoprost was used as the prostamidederivative. The procedures outlined herein can be used to makeencapsulated microparticles of other prostamide derivatives as well.

FIG. 1 is a flow chart illustrating the steps used in the method of thisexample.

As shown in FIG. 1, a first composition was formed by adding 100 mg ofbimatoprost to 20 mL of acetonitrile (CH₃CN) in an Erlenmeyer flask witha magnetic stirrer and stopper. The bimatoprost was solubilized in theacetonitirile. PLGA (900 mg) was added to the solubilized compositionand stirred until the PLGA was solubilized. This first composition canbe understood to be a discontinuous phase or an acetonitrile phasecomposition.

A second composition was formed by combining 800 mL of cottonseed oiland 12.8 mL of Span® 85 in a 1000 mL beaker. Span® 85 is a fatty acidcomposition which comprises oleic acid (C18:1) approx. 74%; linoleicacid (C18:2) approx. 7%; linolenic acid (C18:2) approx. 2%; palmitoleicacid (C16:1) approx. 7%; and palmitic acid (C16:0). Span is a registeredtrademark of ICI Americas, Inc., and the Span® 85 can be obtained frompublic sources, such as Sigma Aldrich. Other Span® products can also beused, such as Span® 80. This composition can be understood to be acontinuous phase or oil-containing composition.

An emulsion was formed by adding the first composition to the secondcomposition. The speed impeller was set at 350 rotations per minute(rpm) to stir the second composition. The first composition was added tothe stirring second composition, and the mixture was allowed to stir for90 minutes.

The emulsion was then evaporated under filter air flow for 45 hours at250 rpm air flow.

Hexane (250 mL) was added to the evaporated product and stirred for 1hour. Subsequently, the suspension was centrifuged at 7000 rpm for 10minutes.

The pellet containing microparticles was rinsed twice by centrifugingwith 100 mL of hexane. The microparticles were resuspended in 15 mL ofhexane.

The microspheres were then dried under filter air flow overnight.

Microspheres were packaged under nitrogen and reduced temperatures tomaintain the temperature at about 5 C. The cooled microspheres weresterilized using gamma radiation (25-35 kGy).

FIGS. 2A, 2B, 2C, 2D, 2E, and 2F illustrate the chemical structure ofbimatoprost (AGN 192024, FIG. 2A), 1513 bimatoprost (FIG. 2B); 5,6-transbimatoprost isomer (FIG. 2C); C1 acid of bimatoprost (FIG. 2D);triphenyphosphine oxide (TPPO; FIG. 2E); and 15-Keto bimatoprost (FIG.2F). Bimatoprost is the active ingredient and the other chemicals areimpurities that may be present. Bimatoprost has a molecular weight of415.6 g/mol, a solubility in water at room temperature of 3.2 mg/mL(which is slightly soluble), and a partition coefficient Log P of 2.4plus or minus 0.1. Bimatoprost is a non-ionizable compound.

The effects of gamma sterilization on the stability of bimatoprost arepresented in the following table (Table 2). The formula abbreviationscorrespond to compounds illustrated in FIGS. 2A-2F.

% wt/wt % wt/wt, % wt/wt % wt/wt Bimatoprost bimatoprost 15-keto 15β5,6-trans none 95.3 1.0 0.2 0.1 sterilized gamma 85.3 2.3 0.1 0.1sterilized

Gamma sterilization appeared to decrease the amount of activebimatoprost by about 10%. Gamma sterilization may also change the colorof the material (e.g., from white to a yellow/brown) and the consistencyof the material (e.g., from a fluffy powder to a more compact powder).

Two separate batches of microparticles were produced using PLGAcopolymers from different suppliers, as presented in Table 3, below

Lactide/Glycolide ratio Batch Random/Block Molecular Inherent SupplierNumber End-groups Weight Viscosity APT 1 75/25   63,200 Da 0.65 dL/gRandom Ester & Hydroxyl BPI 2 75/25 ~97,100 Da 0.67 dL/g Random Ester &Hydroxyl

APT in Table 3 refers to Absorbable Polymer Technologies, and BPI refersto Birmingham Polymers, Inc. The PLGA obtained from APT appeared toprovide better results than PLGA from BPI.

High performance liquid chromatography was used to quantify bimatoprostand impurities present in the microparticles. Analytes were eluted froma Waters Symmetry® C18 reverse-phase column using a mobile phasecomposed of 72/18/10 (water/acetonitrile/methanol v/v/v) containing0.03% (w/v) trifluoroacetic acid. UV detection was performed at 210 nm.Chromatograms of a bimatoprost standard (FIG. 3A), a non-sterilized andsterilized placebo compositions (FIG. 3D and FIG. 3E), andnon-sterilized and sterilized bimatoprost microparticles (FIG. 3B andFIG. 3C) revealed that the microparticles did not contain detectableamounts of impurities as evidenced by the single absorption peak inFIGS. 3A-C.

Particle size was determined by suspending an aliquot of the bimatoprostmicrospheres in 1 mL of deionized water. To this, 100 μL of 10% Tween 80was added. The composition was sonicated for 5 minutes and vortexed for15 seconds. A Coulter LS230 apparatus or equivalent apparatus were usedto measure particle size. Mean particle diameters of tested batches werebetween about 30 μm and about 50 μm. Some particles had diameters up toabout 200 μm. These larger particles were observed in batches that werenot sieved. FIGS. 4A-4D illustrate particle diameters (differentialvolume and number) profile superposition for sterile and non sterileconditions. Each graph illustrates the particle diameter distributionfor a batch of sterile microspheres and non-sterile microspheres. Thedata reveal that sterilization did not significantly impact the particlesize distribution as evidenced by the substantial overlap in thedistribution curves. FIG. 4A illustrates volume % as a function ofparticle diameter for batch DL042 (top left panel), FIG. 4B illustratesnumber % as a function of particle diameter for batch DL042 (top rightpanel), FIG. 4C illustrates volume % as a function of particle diameterfor batch DL043 (bottom left panel), and FIG. 4D illustrates number % asa function of particle diameter for batch DL043 (bottom right panel).

Microscopic aspect was determined using the sample prepared for particlesize determination, described above. Observations were performed atmagnification 5 and 20. FIGS. 5A-5F illustrate shapes of four differentnon-sterile and two different sterile batches of microspheres (FIG. 5Aand FIG. 5B (top two panels) and FIGS. 5C and 5E illustrate non-sterilebatches and FIG. 5D and FIG. 5F (bottom two right panels) illustratesterile batches of microspheres).

The dissolution profile for a batch of microparticles was monitored for28 and 42 days for sterile and non-sterile microspheres using dialysisbags and dissolution media. The dissolution media was phosphate buffer(pH 7.4) and ethanol in a ratio of 90/10. Samples were monitored at 37 Cin a shaker water bath at approximately 110 rpm. At each time point, analiquot of sample was collected and replaced by fresh media. Thedissolution profile for a batch of sterile and non-sterile microspheresis shown in FIG. 6. The dissolution rate for the non-sterilemicroparticles was about 0.7%/day (e.g., 30% released over 42 days). Thedissolution rate for the sterile batch initially appeared to be about0.7%/day and increased after about two weeks. FIG. 6 also shows thatbimatoprost was released faster from gamma sterilized microspheres thannon-sterilized microspheres.

Table 4 below provides information regarding the batches of microspheresprepared in accordance with the present methods and as discussed above.

Batch No. PLGA type/SPAN type Sterilized (Yes/No) DL040 PLGA APT 75/25/No iv 0.65/SPAN 80 DL041 APT/SPAN 85 No DL042 APT/SPAN 80 No DL042APT/SPAN 80 Yes DL043 APT/SPAN 80 No DL043 APT/SPAN 80 Yes

In Table 4, APT refers to the PLGA supplier, as discussed herein, ivrefers to inherent viscosity, and SPAN refers to the fatty acidcomposition described herein, and which is publicly available.

Table 5 below provides information regarding the amount of bimatoprostpresent in non-sterile microspheres. In these microspheres, bimatoprostwas present in an amount of about 5% w/w.

Batch No. Targeted loading % w/w bimatoprost DL040 10% 4.6 DL041 10% 5.1DL042 10% 4.8 DL043 Placebo ND

Batches DL042 and DL043 were gamma sterilized (25-35 kGy) in order toevaluate the stability of the active ingredient when encapsulated withinPLGA. The results are presented in Table 6 below.

Targeted % w/w Batch No. Loading bimatoprost % w/w keto DL042 10% 4.3 NDDL042 sterile 10% 4.2 ND DL043 Placebo ND ND DL043 sterile Placebo ND ND

Example 6 Preparation of Bimatoprost Microspheres

A series of three (3) bimatoprost microsphere (microparticle)formulations, designated Formulations 1, 2 and 3, were prepared asfollows:

Formulations 1 and 2 were prepared using a spray-drying technique.Bimatoprost was dissolved in a solution of a polylactide(co-glycolide)(PLGA) biodegradable polymer and dichloromethane at a theoreticalbimatoprost content of 3 wt %. Different PLGA polymers were used in eachof Formulations 1 and 2. The solution was atomized using a Buchi Model190 spray dryer with an inlet temperature set at 40° C. Thebimatoprost/PLGA polymer microparticles thus formed were recovered fortesting.

Formulation 3 was prepared using a solvent-extraction “continuous”microencapsulation process. The bimatoprost was suspended in a solutionof a PLGA polymer and ethyl acetate at a theoretical bimatoprost contentof 4.3 wt %. The solution was homogenized in the presence of a polyvinylalcohol solution saturated with 4 mg/mL bimatoprost to produce awater-in-oil emulsion. The microparticles were formed upon extraction ofthe solvent in a continuous flow of purified water. The microparticleswere isolated by centrifugation and lyophilized. The dried particleswere then suspended and stirred in purified water at room temperaturefor approximately 2 hours. The particles were again isolated bycentrifugation and lyophilized.

All of the microparticles of Formulations 1, 2 and 3 microparticles wereirradiated with 2.5 Mrad gamma radiation prior to use in the rabbit invivo study, described in Example 8, supra.

Analytical characterization of the microparticles was performed, and theanalytical data are summarized in Table 7.

TABLE 7 Summary of characterization data of Bimatoprost MicroparticleFormulations. Particle Size Bimatoprost Encap- Distribution, ResidualFormulation Content, sulation microns Solvent, Number wt % Efficiencyd10 d90 Mean wt % 1 2.9 96.7 1.8 30.3 14.4 <0.1 2 2.9 96.7 1.4 17.6 8.8<0.1 3 4.0 88.9 2.4 41.4 19.0 0.8

Example 7 In Vitro Testing of Bimatoprost Microspheres

Samples of each of the Example 6 Formulations 1, 2 and 3, bothirradiated with gamma radiation (as set forth in Example 6) andnon-irradiated, were tested in vitro for rate of release of bimatoprost.These in vitro tests were conducted as follows:

Each individual sample of microparticles was placed in a glass vialfilled with receptor medium (9% NaCl in water). To allow for “infinitesink” conditions, the receptor medium volume was chosen so that theconcentration of bimatoprost would never exceed 5% of saturation. Tominimize secondary transport phenomena, e.g. concentration polarizationin the stagnant boundary layer, each of the glass vials was placed intoa shaking water bath at 37° C. Samples were taken for HPLC analysis fromeach vial at defined time points. The HPLC method was as described inUSP 23 (1995) pp. 1791-1798. The concentration values were used tocalculate the cumulative release profiles.

The release profiles are shown in FIG. 7.

As shown in FIG. 7, for each of the samples of Formulations 1 and 2,both irradiated and not irradiated, the release rate of bimatoprost issomewhat faster than the target release rate 7. The samples ofFormulation 3, both irradiated and not irradiated, reasonablyapproximate the target release rate 7.

These data demonstrate that bimatoprost/polymer microparticles can beproduced with different release rates, for example, for use in differentapplications and/or to treat different conditions/diseases. In short,these data demonstrate that bimatoprost/polymer microparticles can beproduced with a range of bimatoprost release profiles as needed to treatdifferent ocular conditions and/or to improve visual acuity.

Example 8 Subconjunctivally Injected Bimatoprost Microspheres

A study of subconjunctival injection in New Zealand white rabbit eyes ofthe Formulation 1, 2 and 3 microparticles of Example 6 was carried out.

The Formulation 1, 2 and 3 microparticles were provided in the form ofdry particulates as six-seven doses per vial. A duplicate set of vialswas supplied for each formulation, dose level, and timepoint (i.e., 20vials for each lot number).

A 2 mg/mL bimatoprost solution (Formulation 4) prepared in salinecontaining 0.1 wt % Tween 80 was provided for dose administration to anyanimals still available following completion of microparticle dosing.

Preparation:

The microparticles in each vial were reconstituted in 2.2 mL ofinjection vehicle. Microparticle/vehicle compositions were usedimmediately upon reconstitution or as soon as possible within 2 hours ofreconstitution. Details of test composition preparation are outlined inTable 8 below.

TABLE 8 No. of Vol. of Bimatoprost vials of Micro- injection SuspensionDose Formulation content 6-7 spheres per vehicle Conc. volume Number (wt%) doses vial (mg) added (mL) (wt %) (mL) 1 2.9 10 168 2.2 7.1 0.15 10336 2.2 13.2 0.15 2 2.9 10 168 2.2 7.1 0.15 10 336 2.2 13.2 0.15 3 3.910 139 2.2 6.2 0.15 10 278 2.2 11.2 0.15 4 2 mg/mL 10 mL none 10 mL 2mg/mL 0.15

The injection vehicle was a sterile, aqueous saline solution containing0.1 wt % Tween 80 (polyoxyethylene-sorbitan monooleate).

To ensure that the appropriate dose of microparticles was injected, thefollowing procedure was used to administer the microparticle doses.

An 18-gauge needle was inserted into a vial of sterile injection vehicleto release the vacuum. An empty syringe was attached to the needle thevial containing the vehicle was inverted, and the appropriate volume(2.2 mL) of vehicle was drawn out. The needle and the syringe waswithdrawn from the vial containing the vehicle, and the needle wasinserted into the vial containing the microparticles to be injected. Thevial of microparticles was vented by inserting a separate needle throughthe septum. Then, leaving this needle in place, the vehicle was injectedinto the vial containing the microparticles, and both needles werewithdrawn.

To suspend the microparticles in the vehicle, the vial was vigorouslyshaken for at least 30 seconds. After the microparticles were welldispersed, the appropriate amount of suspension (0.20 mL) was pulledinto a 1-cc syringe using an 18-gauge needle. The needle was cleared ofany suspension by pulling all of the contents into the syringe.

The 18-gauge needle was replaced with a 25-gauge×⅝-inch needle, and theneedle was purged of air. The microparticles were injected quickly toprevent the microparticles from settling out of solution. Between eachinjection, the microparticles were resuspended by agitating the samplevial.

The entire procedure was repeated as necessary to complete doseadministration. Each vial was used for a maximum of six to seveninjections.

The microparticle doses were stored at −20° C. and then allowed toequilibrate to room temperature for approximately 1-2 hours prior toreconstitution. The injection vehicle was stored at room temperature.

The animals were provided with approximately 150 grams of TekladCertified Hi-Fiber Rabbit Diet daily. The animals were provided tapwater ad libitum.

Prior to placement on study, a physical examination was performed oneach animal. Each animal also had a pre-treatment ophthalmic examination(slit lamp and indirect ophthalmoscopy), performed by a veterinaryophthalmologist.

Prior to placement on study, intraocular pressure (IOP) was determinedfor both eyes of each animal. Proparacaine hydrochloride 0.5% (1-2drops) was delivered to each eye prior to measurements.

Neomycin/Polymyxin/Bacitracin (NPB) Ophthalmic Ointment was placed inboth eyes of each animal once daily on the day prior to injection(Day-1) and two days after injection (Days 2 and 3).

Prior to dosing, the animals were weighed and randomly assigned to 33study groups (Groups A-GG). If any alternate animals remained followingcompletion of microsphere dosing for Groups A-GG, these animals wereassigned to Groups HH, II, and/or JJ.

Animals were anesthetized with an intravenous injection of aketamine/xylazine cocktail (87 mg/mL ketamine, 13 mg/mL xylazine) at avolume of 0.1-0.2 mL/kg. Animals instead were anesthetized with anintramuscular injection of ketamine 100 mg/mL at 35 mg/kg plus xylazine100 mg/mL at 7 mg/kg.

Eyes were prepared for injection as follows: Approximately five minutesprior to injection, eyes were moistened with an ophthalmic Betadinesolution. After five minutes, the Betadine was washed out of the eyeswith sterile saline. Proparacaine hydrochloride 0.5% (1-2 drops) wasdelivered to each eye.

On Day 1, each rabbit in Groups A-GG received a 150-μL, subconjunctivalinjection of the appropriate microparticle formulation into the left eyeusing a 25-gauge×⅝-inch needle. Each rabbit assigned to Groups HH-JJreceived a 150-μL, subconjunctival injection of bimatoprost solutioninto the left eye using a 30-gauge×⅝-inch needle. The bulbar conjunctivain the dorsotemporal quadrant was elevated using forceps. Microparticleformulation or bimatoprost solution was injected into thesubconjunctival space. The actual dose delivered was calculated as adifferential of the syringe weight before and after dosing. Injectionwas performed by board-certified veterinary ophthalmic surgeons.

The animals were observed for mortality/morbidity twice daily.

If an animal was determined to be moribund or under severe distress, theanimal was euthanized with an intravenous injection of commercialeuthanasia solution. Both eyes were explanted, placed in 10% formalin,and stored for possible future evaluation.

The animals were weighed at random, on Day 1, and prior to necropsy.

Intraocular pressure (IOP) was determined for both eyes of eachremaining animal on Days 2, 8±1, 15±1, 22±1, and 29±1. Proparacainehydrochloride 0.5% (1-2 drops) was delivered to each eye prior tomeasurements. IOP was evaluated with a Medtronic Solan, Model 30 classicpneumatonometer on conditioned rabbits. A three-point diurnal curve wasestablished: Intraocular pressure was recorded at 8 a.m., 12 p.m., and 4p.m., with a ±1 hour range for each of these times.

Gross ocular observations were performed once weekly.

Ophthalmic observations (slit lamp and indirect ophthalmoscopy) wereperformed on both eyes of each remaining animal on Days 2, 8±1, 15±1,22±1, and 29±1. Observations were performed by the same veterinaryophthalmologist for all timepoints.

Blood (at least 0.5 mL) was collected from each animal via ear vein orcardiac puncture prior to euthanasia. Animals were weighed andanesthetized with an intravenous injection of a ketamine/xylazinecocktail (87 mg/mL ketamine, 13 mg/mL xylazine) at a volume of 0.1mL/kg. Animals were euthanized with an intravenous injection ofcommercial euthanasia solution following blood collection.

Each approximate 0.5-mL blood sample was collected into a single,pre-labeled K₃ EDTA tube. Each tube was inverted 5-10 times to ensureadequate mixing of blood and anticoagulant. The tubes were then placedon ice. Using a glass pipette, duplicate 0.2-mL aliquots of whole bloodwere transferred into silanized round bottom 13×100 mm screw top glasscentrifuge tubes with Teflon caps containing 2.5 mL of 50%acetonitrile/50% methanol (high purity). Tubes were then vortexed. Bloodsamples were stored temporarily on wet ice until transferred to afreezer (at or below −20° C.) Samples were maintained at or below −20°C. until submitted for testing.

For eyes designated for pharmacokinetics analysis, aqueous humor sampleswere collected from all eyes before the eyes were enucleated, and thevolume collected was measured and recorded. Both globes were enucleatedand frozen in liquid nitrogen (at −20° C. or below).

Eyes were dissected as follows: The iris/ciliary body (ICB) wascollected first, followed by collection of the vitreous humor (VH) andthe retina.

Tissue weights were recorded for all tissues collected from all eyes.Careful attention was used to ensure that there is no crosscontamination during dissection.

Aqueous humor (AH) and VH were placed in silanized amber screw-cap glassvials (3 mL) with Teflon or other inert washer. ICB and retina sampleswere placed in silanized round bottom 13×100 mm screw top glasscentrifuge tubes with Teflon caps. Collected tissues were frozen on dryice immediately after collection. All samples were stored at −20° C. orbelow until analysis.

The acceptable time ranges for sample collections (blood and oculartissues) are summarized in the following Table 9:

TABLE 9 Scheduled Collection Time (Day) Acceptable Time Range 2 ±0 days8, 15, 22, 29 ±1 day

Ocular tissue samples collected for pharmacokinetics were analyzed usinga validated LC-MS/MS method. Samples were quantified with AH, ICB, VHand retina assay range for bimatoprost and a bimatoprost metabolite of0.1 ng or ng/mL. Assay range for blood was 0.125 and 0.25 ng/mL forbimatoprost and the bimatoprost metabolite, respectively.

Following subconjunctival injection of the microparticles, mainly thebimatoprost metabolite was detected in the AH, and bimatoprost wasdetected in blood. Both bimatoprost and the bimatoprost metabolite weredetected in the ICB, VH and retina. Contralateral diffusion ofbimatoprost and the bimatoprost metabolite to the untreated eyes wasdetected in the ICB and VH. In contrast, no drug levels were detected inall tissues at all timepoints (Day 1-14) following subconjunctivalinjection of 300 μg bimatoprost solution. Significantly, thesubconjunctival bimatoprost microparticles caused little or no eyeredness (hyperemia) in the rabbits. This is an unexpected advantage, forexample, since topical administration of bimatoprost-containing eyedrops can cause instances of hyperemia.

For all formulations dosed either at 300 μg or 600 μg dose, AHbimatoprost levels were detected at below the limit of quantification(BLQ) (<0.1 ng/mL) for all timepoints. Similar levels were detected forthe bimatoprost metabolite, with the exception of a few treated eyeswhich had an AH level slightly above the BLQ level.

For all formulations dosed either at 300 μg or 600 μg dose, bimatoprostand bimatoprost metabolite were detected in the ICB at the earliesttimepoint of Day 1. Only one formulation resulted in drug levels beyondDay 1 up to Day 21.

For all formulations dosed either at 300 μg or 600 μg dose, bimatoprostwas detected in the VH at the earliest timepoint of about Day 1. Onlyone formulation, Formulation 3, resulted in drug levels beyond Day 1 upto Day 21. The bimatoprost VH maximum concentration for this formulationdosed at 300 μg and 600 μg was 1.73 ng/mL (Day 1) and 13.4 ng/mL (Day1), respectively.

For all formulations dosed either at 300 μg or 600 μg dose, bimatoprostand bimatoprost metabolite were detected in the retina at the earliesttimepoint of Day 1. Only one formulation resulted in bimatoprost levelsbeyond Day 1 up to Day 7, Formulation 3 (300 μg) and beyond Day 1 up toDay 21, Formulation 3 (600 μg). The a bimatoprost retinal maximumconcentration for this formulation dosed at 300 μg and 600 μgbimatoprost was 101 ng/g (Day 7) and 289 ng/g (Day 1), respectively.

For all formulations administered at either 300 μg or 600 μg dose, blooddrug levels were mainly at BLQ.

No mortality or drug-related effects on ophthalmic observations, bodyweight, or gross ocular observations were observed up to 1 month afteradministration.

This study shows that bimatoprost microparticles can be administeredsubconjunctively to produce concentrations, for example therapeuticallyeffective concentrations, of bimatoprost/metabolite in various portionsof the eye for periods of time of at least about 1 week or at leastabout 2 weeks or at least about 3 weeks. Thus, these subconjunctivalinjected bimatoprost microspheres can be used to effectively treatglaucoma. The fact that a relatively large amount of the bimatoprostadministered subconjunctivally was passed to the posterior chamber, asbimatoprost or the bimatoprost metabolite is significant, for example,in treating conditions/diseases of the retina, for example, dryage-related macular degeneration, as well as other retinalconditions/diseases.

Example 9 Subconjunctivally Injected Bimatoprost Microspheres to TreatGlaucoma

A 56 year old male suffering from glaucoma in both eyes receives asubconjunctival injection of microspheres containing bimatoprost and acombination of a PLA and PLGA within the subconjunctival space of eacheye. The microspheres contain 4% bimatoprost, and 40 mg of microspheresare used for each injection. In about two days, the patient reports asubstantial relief in ocular comfort. Determination of intraocularpressure (TOP) reveals that the IOP has decreased in each eye, theaverage intraocular pressure measured at 8:00 AM has decreased from 28mm Hg to 14.3 mm Hg. The patient is monitored every other day for about1 month. Intraocular pressure levels remain below 15 mm Hg for thisperiod of time, and the patient reports reduced ocular discomfort.Little or no ocular hyperemia is observed.

Example 10 Oil in Water Method for Making Bimatoprost Microspheres

This example describes an oil in water emulsion evaporation for makingmicroparticles that include a prostamide derivative encapsulated by abiodegradable polymer. In the specific example, bimatoprost was used asthe prostamide derivative. The procedures outlined herein can be used tomake encapsulated microparticles of other prostamide derivatives aswell.

Preparation of bimatoprost containing microspheres is difficult due to alack of a significant solubility differential of bimatoprost in water aswell as in many organic phases such as ethyl acetate and methylenechloride. When known oil in water emulsion evaporation processes areused most of the bimatoprost escapes into the aqueous phase. ThisExample sets forth an oil in water emulsion evaporation process whichhas been successfully used to make bimatoprost containing PLGAmicrospheres. An oil in water process is used in which an excess amountof the bimatoprost is added to the aqueous phase so as to saturate theaqueous phase to thereby prevent or reduce escape of the bimatoprostfrom the aqueous phase, with the result of higher drug loading (up toabout 10 wt %) in the microspheres made.

The following process was use to make biodegradable bimatoprostmicrospheres using Poly(DL-lactide-co-glycolide) (PLGA). Anemulsion/solvent evaporation technique was used. The continuous oraqueous non-solvent phase was saturated with bimatoprost to prevent lossof bimatoprost from the polymer phase and increase loading efficiency.As drug diffuses away from the polymer phase during microspherehardening counter-diffusion of drug into the polymer phase from thesaturated aqueous phase minimizes loss of the bimatoprost.

Bimatoprost loaded PLGA microspheres were prepared using theemulsion/evaporation procedure as follows: (1) 1 gram of PLGA (75:25)was combined with 1 gram of bimatoprost in 60 milliliters ofdichloromethane (an organic solvent) (this formed the polymer phase;also referred to as the oil phase or as the organic phase); (2) 10 gramsof polyvinyl alcohol (“PVA”) was combined with 1 gram of bimatoprost in300 milliliters of water to thereby form a saturated aqueous phase(aqueous phase); (3) the polymer phase was emulsified with the aqueousphase by drop wise addition of the polymer phase into the aqueous phaseunder continuous mixing with a high shear impeller; (4) the resultingPLGA microspheres were hardened through the evaporation of thedichloromethane by stirring the emulsion in an open beaker for 24 hours;(5) the hardened microspheres were separated from the remaining PVAsolution by centrifugation; (6) the microsphere pellets were suspendedand washed 3 times with water; (7) the microspheres were dried undervacuum at 45° C. overnight; (8) the dried microspheres were sized to 38microns through sieves.

The percent of bimatoprost loaded into the microspheres by this processwas analyzed by HPLC to be 11.91%. The particle size distribution of themicrospheres was determined to have a mean of 8.7 microns. The percentrelease of bimatoprost from the microspheres was assessed usingdialysis.

In variants of this process the solvent used can be, for example, ethylacetate or methylene chloride. The PLGA polymer used can be, forexample, 50:50 DL-PLG acid, 65:35 DL-PLG, 65:35 COOH, 75:25 COOH or75:25 laurl ester capped. DL-PLG acid 50:50 DL-PLG acid. The meanmicroparticle diameter can be from about 5 microns and about 20 microns.

In another variant of our an oil in water emulsion evaporation processfor making microparticles that include a prostamide derivativeencapsulated by a biodegradable polymer, a solid bimatoprost can bepresent in the emulsion during preparation. In such a variant of ourprocess, the drug (bimatoprost) concentration exceeds its solubilitiesin both the aqueous and the organic phases and the drug is present asmicrofine particles in both phases as well. As the PLGA hardens when theorganic phase evaporates, the drug can be captured as microfineparticles as well as from precipitation.

Significantly, bimatoprost encapsulation within PLGA allows terminalsterilization with gamma radiation (i.e. by exposing the microparticlesto gamma radiation at a dose of about 25-35 kGy) with low to moderateimpact on the biological stability of the encapsulated bimatoprost.

All references, articles, publications and patents and patentapplications cited herein are incorporated by reference in theirentireties.

While this invention has been described with respect to various specificexamples and embodiments, it is to be understood that the invention isnot limited thereto and that it can be variously practiced within thescope of the following claims.

We claim:
 1. A population of microparticles comprising a polymericcomponent encapsulating a prostamide component in the form ofoil-in-water emulsified microparticles.
 2. The population of claim 1,wherein the polymeric component comprises a poly (lactide-co-glycolide)copolymer, and the prostamide component comprises at least oneprostamide derivative selected from the group consisting of bimatoprost,salts thereof, and mixtures thereof.
 3. The population of claim 1 whichis terminally sterilized.
 4. The population of claim 3, wherein at least80% of the prostamide component remains stable after the sterilization.5. The population of claim 1 having a mean particle diameter from about30 μm to about 50 μm.
 6. The population of claim 1, wherein the maximumparticle diameter is less than about 200 μm.
 7. The population of claim1, wherein the microparticles have a release rate of the prostamidecomponent of about 0.7% per day in vitro.
 8. The population of claim 1,wherein the prostamide component comprises about 10% wt/wt of themicroparticles.
 9. The population of claim 1, wherein the prostamidecomponent comprises about 5% wt/wt of the microparticles.